The negatively charged aminophospholipid, phosphatidylserine (PtdSer), is located in the inner leaflet of the plasma membrane in normal cells, and may be exposed to the outer leaflet under some immune and blood coagulation processes. Meanwhile, Ptdser exposed to apoptotic cells can be recognized and eliminated by various immune cells, whereas on the surface of activated platelets Ptdser interacts with coagulation factors prompting enhanced production of thrombin which significantly facilitates blood coagulation. In the case where PtdSer fails in exposure or mistakenly occurs, there are occurrences of certain immunological and haematological diseases, such as the Scott syndrome and Systemic lupus erythematosus. Besides, viruses , Ebola virus (EBOV)) can invade host cells through binding the exposed PtdSer. Most recently, the Corona Virus Disease 2019 (COVID-19) has been similarly linked to PtdSer or its receptors. Therefore, it is essential to comprehensively understand PtdSer and its functional characteristics. Therefore, this review summarizes Ptdser, its eversion mechanism; interaction mechanism, particularly with its immune receptors and coagulation factors; recognition sites; and its function in immune and blood processes. This review illustrates the potential aspects for the underlying pathogenic mechanism of PtdSer-related diseases, and the discovery of new therapeutic strategies as well. In eukaryotic cells, the distribution of lipids on biological membranes is asymmetric . GlycospApoptosis  is also 2+ or caspase ..193].(ii)Antithrombin (AT) is an enzyme that inhibits the coagulation system, which is a serine protease inhibitor (serpin) . The phy(iii)2+-dependent manner, lead to the decomposition and inactivation of the Tenase complex and Prothrombinase complex, and inhibit blood coagulation.Another anticoagulant pathway is the PtdSer-dependent APC anticoagulant system. It inhibits the activities of FVIIIa and FVa Fig.  b. In otDuring the coagulation process, after platelet activation, the content of PtdSer on the outer leaflets of platelets increases significantly. Tenase complex and Prothrombinase complex can interact with PtdSer to promote thrombin generation . During t manner . FVIIIa tivation . The ligtivation . Howevertion [2+ . There a2+-depend manner and participate in the process of coagulation and anticoagulation [Proteins FVII, FIX, FX, Prothrombin APC have a GLA domain at the N-terminal, which can bind to PtdSer in a Cagulation . In thisgulation . So thesgulation . Also, igulation , 97–100.The binding of the GLA domain to the PtdSer-exposed membrane is mediated by multiple interactions: Calcium coordination, ions, van der Waals forces, and hydrophobic interactions . GLA dom2+ ions. Researches have established an atomic model of membrane binding GLA domains [2+ ions have two roles in the GLA domain: The four internal Ca2+ ions are mainly responsible for the correct folding of the GLA domain to insert into the membrane; the external Ca2+ ions anchor proteins on the membrane by directly contacting lipids. This model further indicates that Ca2+ is necessary for the GLA domain to bind to the membrane. Studies have shown that the GLA domain interacts with the negatively charged head group of the membrane phospholipid [2+ in the GLA domain to performing their function. Furthermore, the stoichiometric ratio of the only complete PtdSer-GLA domain complex whose structure is determined by X-ray crystallography is 1:1, that in other words, one PtdSer molecule binding to one GLA domain [The GLA domain’s function is highly dependent on Ca domains , showingpholipid , 200. MoA domain .Additionally, PROS1, one of the many coagulation factors consumed during the vigorous blood coagulation process, is a vitamin K-dependent anticoagulant protein as well , 105. It2063 and Trp2064) in this lipid-binding pocket. Mutating these Trps eliminates the binding of FV to the membrane, indicating that these two Trp residues are necessary for the binding of FVa to PtdSer [2313-His2315 fragment of the C2 domain of FVIII is involved in membrane binding [The PtdSer recognition site of FVa and VIIIa is located in their N-terminal C2 domain , 202. Tho PtdSer . Some reo PtdSer  and the  binding . All theIn recent years, with the development of the coagulation mechanism, more and more diseases, such as essential hypertension (EH) , HemophiThe PtdSer exposed on the surface of platelets facilitates the assembly of Tenase complex and Prothrombinase complex, thereby promoting the production of thrombin on the surface of activated platelets . FurtherThrombosis is the main cause of death in patients with EH . StudiesHemophilia A is a genetic disease. It is a serious bleeding disorder caused by mutations in the FVIII ) gene on the X chromosome . StudiesRecent studies have also found that PtdSer, exposed on the surface of some mesenchymal stem cells and their derived extracellular vesicles, potentiates blood clot formation. While the use of annexin V to block PtdSer on their surface will lead to their procoagulant activity partially lost . Also, s2+. However, the relatively large choline group of PtdCho will hinder the binding of the PtdCho and GLA domain [Both GLA domain , 218 andA domain .2+-dependent manner, thereby supports neuronal survival and differentiation in neuronal signal transduction [Discoidin-like C2 domain not only appears in coagulation factors (FV and FVIII) , 202 butsduction , 223. Hosduction , 225.2+ dependent. It could be speculated that whether other proteins containing IgV-like domains also have the property of binding to PtdSer, which needs further study.There are IgV-like domains in some immune receptor proteins, like TIM family proteins , CD300 f2+ [etc [Besides these Ptdser recognition sites mentioned above Table , there a2+ . Annexin2+ –230. In 2+ . TSP-1 c2+ . Besides2+ , 68 in e2+ , 165, ce2+ [etc .Table 2Corona Virus Disease 2019 (COVID-19) is a viral infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which poses a major challenge to the World Health Organization . Acute iStudies have speculated that PtdSer exposure may be involved in the SARS-CoV-2 infection of the outer leaflets of the cell membrane. PtdSer may be a potential mechanism or participant of inflammation and coagulation abnormalities in COVID-19 patients , 24. As Similarly, some researchers  have shoBesides PtdSer, it was speculated that PROS1 might mechanically associate excessive blood coagulation with immune response in COVID-19 . PROS1 c12, can use the PtdSer exposed on their surface to bind to TIM to enter host cells. Therefore, we suggested that therapeutic targeting for TIM-1 can prevent and/or treat COVID-19 and prevent the above virus from infected.It has also been reported that the receptor for PtdSer, TIM-1 (KIM-1), may be the receptor for SARS-CoV-2 in the lung and kidney . COVID-1Based on various studies , 239, it2+-dependent APC anticoagulant system [Immune systems and blood coagulation are vital for basic physiological functions. When tissues are injured, hemostasis is always the first step to control the injury. The hemostasis process usually requires platelets to complete. The cell-based coagulation mechanism has been explained in this article (section The Cell-Based Model of Hemostasis involving PtdSer). Blood coagulation and anticoagulation are very complicated processes involving the participation of various factors, and PtdSer is indispensable for activated platelets to produce thrombin –32, 103.t system , 196. Th(i)PtdSer is exposed on the surface of apoptotic cells. The exposure of PtdSer requires the activation of scramblase Xkr8 and the inactivation of flippase ATP11C and ATP11A. Caspase3/7 participates in the activation and the inactivation –44.(ii)PtdSer receptors on macrophages can recognize and bind PtdSer normally. There are many PtdSer receptors on macrophages, including TAM receptor family  , TIM rec(iii)PtdSer receptors can successfully activate downstream signal pathways after binding to PtdSer. For instance, the binding of TAM to PtdSer can activate Rac1 to activate phagocytosis  and inhiImmune system is used to eliminate foreign antigens, or damaged cells, apoptotic cells, and tumor cells produced in our body to maintain homeostasis . In this2+ level in the activated platelets increases, which leads to the activation of scramblase TMEM16F and the inactivation of flippase ATP11C and ATP11A, thereby causing PtdSer exposure on the platelet cell membrane. Then, PtdSer binding to various coagulation factors  on the surface of activated platelets will promote their enzymatic activity and causes a burst of thrombin production. (iii) A large amount of thrombin can activate Fibrinogen (FI) and FXIII (FXIII) to generate Fibrin (FIa) and FXIIIa. FXIIIa can cross-link with FIa to form a Cross-linked fibrin clot, which is very important for the formation of a blood clot at the injured site. The formation of blood clots can prevent excessive bleeding [The coagulation process which PtdSer participated in can be briefly summarized as follows: (i) When tissue cells are injured, a little thrombin will be produced. (ii) A small amount of thrombin can activate platelets, and the Cableeding , 179.The successful elimination of apoptotic cells is closely dependent on each of the above parts. Particular diseases will occur due to the absence of individual steps. For example, PtdSer exposure failure may be the cause of SLE . ApoptotBesides eliminating apoptotic cells, the binding of PtdSer to immune cell receptors can lead to other immune functions. For example, inhibiting the expression of inflammatory factors and NF-κB inhibit inflammation , 88. TheHowever, some viruses  that recognize and bind to PtdSer, such as GLA domain, discoidin-like C2 domain, IgV-like domain, etc. It is not excluded that other coagulation-related factors or immune receptors that have these PtdSer binding sites may also have the property of binding to PtdSer. More researches are needed and discovery to verify this hypothesis.Since the coagulation and immune mechanisms have been continuously improved in recent years, many factors or receptors involved have been found. The relationship between the coagulation and immune mechanisms is being established , 26. ThiPtdSer belongs to phospholipids on the membrane, and its importance for the biological membrane is obvious. PtdSer exposure is of great significance for blood coagulation –32, 103


The aim of this study was to compare fluoride release from four selected fissure sealants: fissure sealant manufactured by Arkona, Helioseal F, Helioseal F Plus, and Conseal. Tested parameters included emission of fluoride ions into saline (0.9% NaCl) and deionized water over a period of 2 weeks. Values were recorded after 1, 3, 24, 48, 72, and 96 h, and then after 1 and 2 weeks. All sealants were characterized by a constant fluoride release level. The highest fluoride release level was noted for Conseal (0.0169 ppm/mg), while the lowest was noted for fissure sealant manufactured by Arkona (0.063 ppm/mg). Fissure sealants, which contain fluoride, release it not only during polymerization, but also for several days after application. The use of fissure sealants whose composition includes fluoride is an effective method of preventing tooth decay. Tooth decay is one of the most common oral diseases. It occurs in all people, regardless of race, gender, or age, and is therefore classified as a lifestyle disease ,2.For years, it has been known that the best way to fight this disease is to prevent it; for this reason, dentistry employs a whole range of preventive procedures aimed at inhibiting the development of tooth decay. One of these is the use of the most cariostatic of elements, i.e., fluorine. Fluorine compounds have been playing a major role in tooth disease prevention for 70 years and are the most effective method of combating tooth decay .Fluorine is one of the most common elements found in the environment. Due to its properties, it is widely used in medicine and dentistry as well as industry and agriculture.As a highly bioactive element, it affects a number of processes in living organisms. Fluoride ions are inhibitors or, less frequently, activators of multiple enzymes; they affect protein biosynthesis as well as carbohydrate and lipid metabolism, which enables them to modify certain biological functions of living organisms ,4,5,6.Dentistry, however, makes use of their anticariogenic action—fluoride deposits itself in the enamel and reduces the production of acid by plaque bacteria, thus inhibiting demineralization and increasing remineralization ,7.Fluoride is being more and more frequently added to filling materials, including sealants; this provides a reserve of fluoride ions for hard dental tissues through gradual release over a longer period of time . It has The aim of this study was to compare fluoride release from four selected fissure sealants—fissure sealant manufactured by Arkona, Helioseal F, Helioseal F Plus and Conseal—into two different media: deionized water and saline.Four different fissure sealants were used in the study: Helioseal F (Ivoclar Vivadent), Helioseal F Plus (Ivoclar Vivadent), Conseal F (SDI), and fissure sealant manufactured by Arkona. Ten disc-shaped samples with a diameter of 4 mm and a height of 2 mm were made from each material. As per recommendations of the manufacturers, samples were cured using a Light Pro (GC) lamp for 20 s. The samples were then immersed in deionized water  and in NaCl solution  with a temperature of 37 °C.An ORION 9609 ion-selective electrode, coupled with a CP-551 ELMETRON microcomputer pH/ion meter, calibrated before each measurement, was used to measure fluoride ion emission.Fluoride release was checked after 1, 3, 24, 48, 72, and 96 h, and then after 1 and 2 weeks.X-ray Diffraction (XRD) measurements were made on the X’Pert PRO X-ray diffractometer  by PANalytical. Scanning electron microscope (SEM) micrographs were made on FEI Nova NanoSEM 230 microscope. Fourier Transform Infrared (FTIR) spectra measurements were performed on a Thermo Scientific Nicolet iS50 FTIR spectrometer equipped with an ATR module (iS50 ATR). The source of infrared radiation was a HeNe laser.p-Values of < 0.05 were deemed statistically significant. Statistical analyses were performed using Statistica v.13.3 .Descriptive statistics, including mean standard deviation (±SD) and 95% confidence interval (±95% CI), were calculated for each of the four studied groups. Data distribution was performed using the Shapiro–Wilk test of normality. ANOVA for dependent samples was used to determine if there were significant differences in fluoride ion release for each material during the fluoride ion release periods. ANOVA for independent groups was used to calculate the differences between the studied groups. A post-hoc Tukey’s test was performed to compare the studied groups. 3 crystalline phases. Their origin is from 7% submicron admixture, from which this material is made.3 phases identified by XRD measurements are too small to be visible in this magnification. After release of fluoride ions, the surface of each sample become more porous and, for the Conseal sample, deep channels are visible both for release into deionized water and saline solution. Moreover, some crystalline structures  are visible for all samples after release in saline solution.SEM images of four different kinds of fissure sealants are presented in −1 originate from the vibrations of the C=O double bond, the band at 1250 cm−1 is related to the vibration of the C–O–C molecule, the band located at 1610 cm−1 corresponds to the characteristic for dental materials C=C double bond, while intense bands at 2866 and 2960 cm−1 are related to the vibrations of the C-H bond. Since all materials contain fluoride, there is also a visible peak at 749.7 cm−1 for the F–OH–F configuration. Moreover, the OH− group could be substituted in two positions (F–OH–F or F–HO–F) and might be located from the one to the another with relatively low energy [−1 associated with the vibration of the O–H group.p = 0.0005, p < 0.0001 and p = 0.0034). Arkona exhibited the highest fluoride release level at 72 h of incubation, Helioseal at 24 h of incubation, Conseal at 1 h of incubation, and Helioseal Plus at between 1 and 3 h of incubation. As shown in Emission of fluoride ions from the studied fissure sealants manufactured by Arkona, Helioseal, Conseal and Helioseal Plus—into a saline solution is shown in p < 0.0001 for all).p < 0.0004), Conseal (p < 0.0001), and Helioseal Plus (p = 0.0001). The highest fluoride ion release for Arkona was recorded at 48 h of incubation, for Helioseal at 2 weeks of incubation, for Conseal at 1 h of incubation, and for Helioseal Plus at 3 h of incubation. p < 0.001 for all).There were significant differences between the mean fluoride release levels from the fissure sealant manufactured by Arkona, Helioseal, Conseal, and Helioseal Plus . Signifip < 0.0001).Systematic preventive procedures performed at the dentist’s office are an important part of the fight against tooth decay. They are also the basic element of difficult interdisciplinary dental treatment, especially in children with disabilities as well as in patients with systemic diseases ,12,13,14Sealants are liquid fillings which are not permanent, but effectively protect the tooth by releasing fluoride. As early as the 1930s, fluoride was shown to significantly reduce the severity of caries .Fluorine is a cyclical element that is very common in nature. It is the thirteenth most abundant element in the Earth’s crust and is a constituent of multiple minerals, e.g., fluorite, cryolite, fluorapatite, and topaz.Fluorine is also classified as a trace element and is present in mammals at 500 mg/kg of dry mass .Daily fluoride intake requirements of the human body vary with age. According to Moody , fluorid−) exchange in hydroxyapatite. Electrostatically, the interaction of calcium and fluoride (F−) ions is greater than that of Ca2+ and OH− ions, which ensures higher stability of the crystal lattice and, consequently, lower solubility of apatite in acids. These data provide an explanation for the origin of the long-standing principle of fluoride action, which boils down to “fluorides make teeth more resistant to caries” [Fluorine is one of the most electronegative elements and, in ionized form, has a strong affinity for hydroxyl ion  exhibit a higher degree of fluoride ion emission into saline, although the difference was statistically significant only for Helioseal F. The fissure sealant manufactured by Arkona released the same number of fluoride ions into both media.Results of our study are, therefore, inconsistent with results obtained by most authors. Most studies indicate that fluoride release is greater in deionized water than in artificial and human saliva or a saline solution ,35,36,37In their study of materials, Rezk-Lega et al.  and MallTesting indicated statistically significant differences in fluoride release over the given time periods for all materials in each medium.For the Conseal sealant, the highest fluoride release level was noted after only one hour of incubation, both in deionized water and in saline, while, for the Helioseal F Plus sealant, the highest level was observed after 3 h for both media.The other two sealants exhibited significant differences in the timing of the highest ion release level.For Helioseal F, the highest fluoride emission was noted at 24 h of incubation in saline, compared to 2 weeks for deionized water, while the fissure sealant manufactured by Arkona showed the highest emission at 72 h of incubation in saline and 48 h of incubation in deionized water.These results are consistent with results obtained by other researchers ,43,44,45When assessing fluoride release from giomers and fissure-sealing resins, Sismanoglu  noted thAll prepared samples have polymerized properly, which was confirmed by existence of amorphic phases in the XRD diagram and in typical bands for dental materials in the FT-IR spectra.Fissure sealants, which contain fluoride, release it not only during polymerization, but also for several days after application. Further studies are needed to determine the long-term capacity of sealants to release fluoride.Each of the fissure sealants released the largest number of fluoride ions in a different time interval. The highest mean fluoride release level was observed for Conseal sealant after placing samples into saline solution as well as into deionized water. The above material also showed the highest cumulative fluoride ion release into the both solutions.The use of fissure sealants whose composition includes fluoride is an effective method of preventing tooth decay.


RLRs, a major component in antiviral immune recognition. By annotating RLRs in the genomes of 58 representative species across metazoan evolution, we explored the gene expansion of RLRs in Lophotrochozoa. Of note, the N-terminal domains of lophotrochozoan RLRs showed the most striking diversity which evolved independently by domain grafting. Exon–intron structures were revealed to be prevalent in the domain grafting of lophotrochozoan RLRs based on an analysis of sibling paralogs and orthologs. In more than half of the cases, the mechanism of ‘exonization/pseudoexonization’ led to the generation of non-canonical N-terminal domains. Transcriptomic studies revealed that many non-canonical RLRs display immune-related expression patterns. Two of these RLRs showed obvious evidence of positive selection, which may be the result of host defense selection pressure. Overall, our study suggests that the complex and unique domain arrangement of lophotrochozoan RLRs might result from domain grafting, exon–intron divergence, expression diversification, and positive selection, which may have led to functionally distinct lophotrochozoan RLRs.Invertebrates do not possess adaptive immunity but have evolved a variety of unique repertoires of innate immune sensors. In this study, we explored the immune diversity and specificity of invertebrates based on the lophotrochozoan  The immune system has long been known for its remarkably rapid evolution due to strong selective drivers, such as fast-evolving pathogens ,2. LophoNLRs), membrane-bound Toll-like receptors (TLRs), C-type lectin receptors (CLRs), scavenger receptors, AIM2-like receptors (ALRs), and RIG-I-like receptors (RLRs) [Animals distinguish self from non-self by using a variety of PRRs ,12. PRRss (RLRs) .RLRs are a family of three DExD/H box-containing RNA helicases [RLRs include retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2) [MDA5 and RIG-I contain two N-terminal caspase (cysteine-dependent aspartate-specific proteases) recruitment domains (CARDs), followed by an intermediate DEAD/DEAH box helicase domain (DEXDc + HELICc domains) and a C-terminal RIG-I_C-RD domain [LGP2 lacks CARDs and retains the DEAD/DEAH box helicase domain and RIG-I_C-RD domain. The simultaneous presence of CARD and RNA helicase domains in a single molecule, as observed in MDA5 and RIG-I, is unique [RLRs in early animals differs from that in vertebrates. Four types of structural compositions of RLR proteins are found in the brachiopod Lingula anatina [LanRLRs presents different domains, including a death effector domain (DED) and caspase catalytic (CASc) domains, in addition to CARD. Only one CARD has been found at the N-terminus of the RLRs in cnidarians [RLRs across all metazoans is unknown, especially the evolutionary pattern of their domain architecture.elicases . In vert2 (LGP2) . MDA5 anD domain . Helicass unique . Accordis unique . Recent  anatina . The N-tidarians . HoweverRLRs are responsible for the intracellular dsRNA sensor induced by type I IFN [MDA5 or RIG-I knockout mice revealed that this TLR-independent pathway is central to innate immunity against viral infection [MDA5 and RIG-I are IFN-stimulated genes, a positive feedback loop that generates a potent antiviral state is established [RLRs contain three types of functional domains: the two CARD domains at the N-terminus that are responsible for downstream signaling transduction, the DEXDc domain and the HELICc domain in the center that are responsible for RNA recognition, and the C-terminal RIG-I_C-RD domain that assists in pathogen recognition by binding to specific viral RNAs [MDA-5 and RIG-I interact with the CARD domain of the mitochondrial protein IFN-β promoter stimulator-1 . Thereafter, TNF-receptor-associated factor-3 (TRAF-3) is recruited, and TRAF family member-associated NF-κB-activator binding kinase-1 (TBK1) and inducible IκB kinase (IKK) are activated [IRF-3 and IRF-7 and activate NF-κB, which translocates to the nucleus to induce type I IFN expression [MAVS-dependent RLR signaling has been shown to be involved in viral immunity in mollusks [LGP2 lacks CARDs, it does not trigger immune responses; however, it can regulate RIG-I and MDA5 signaling [LGP2 is attributed to its retained helicase domain and RD [As one of the main cytoplasmic PRRs, pe I IFN ,24. An anfection ,26,27. Mablished ,29. The ral RNAs . MDA-5 activated ,32,33. Tpression . MAVS-demollusks . Furtherignaling ,37. The n and RD ,39. RLRs emerge, and their functional diversity. Previous studies suggested that full-length RLRs are a vertebrate-specific evolutionary novelty, although the building blocks of RLRs may have been present in closely related prevertebrate animals [RLRs present in mammals; however, the studies ultimately concluded that RLR evolution was driven by a complex series of domain grafting and gene fusion events in CARD domains. Based on recent studies, the RLR-based immune system is not vertebrate-specific but originates in the earliest multicellular animals [RLRs functionally diversified through a series of gene duplication events followed by protein coding changes, which modulate the RNA-binding properties of different RLRs by altering key contact residues within the C-terminal RD [RLRs are involved in a long-term evolutionary arms race with viral RNA molecules, suggesting that the structure of viral RNA may have shaped the evolution of animal innate immunity [RLRs, these receptors (and/or their building blocks) have been reported to originate early in metazoan evolution [RLR genes in many species. For example, most mammals possess RIG-I; however, RIG-I is lost in at least one mammalian species, the Chinese tree shrew [RIG-I, both MDA5 and LGP2 have undergone strong positive selection in Chinese tree shrews, and positively selected sites in MDA5 endowed the substitute function for the lost RIG-I [RLR gene family in reptiles, birds, and other chordates also revealed that purification selection was dominant [Evolutionary studies have painted a complex picture of how  animals ,40. Thes animals . RLRs fuminal RD . There iimmunity . Althougvolution ,42 and, ee shrew . Interesst RIG-I . Adaptivdominant ,46. HoweRLR gene family in lophotrochozoans. Briefly, we annotated 227 RLR genes from 58 species across the metazoan phylogeny, with emphasis on the molecular evolutionary dynamics of RLRs in lophotrochozoans. Further, we explored their phylogeny and domain composition, which revealed that lophotrochozoan RLRs exhibited highly diverse and complex N-terminal domain integration. We also established an exon–intron structure to investigate the molecular mechanism underlying domain grafting. Evolutionary selection signals and tissue and infective gene expression levels were also calculated. Overall, a comprehensive molecular evolution analysis of the RLR gene family, which is not only associated with rapid domain grafting but also with the potential for an immune response by positive selection, was carried out.Here, we explored the immune diversity and specificity of the RLR gene family across all metazoans, we first annotated 227 RLRs in the genomes of 58 representative species with different evolutionary positions (RLR genes were identified in the sponge of the phylum Porifera (Amphimedon queenslandica), suggesting potential origins of RLRs in early metazoans (RLR gene was completely lost in arthropods of the 58 species investigated (RLRs in most (88%) of the chordates were annotated, while seven RLRs were annotated in Branchiostoma floridae. Compared with chordate animals, RLRs were extensively expanded in most (60%) lophotrochozoans. Further, 13 RLRs in the genome of Crassostrea gigas, 12 in Bathymodiolus platifrons, and 19 in Mytilus coruscus were predicted. The expansion of RLRs was also identified in the echinoderm Strongylocentrotus purpuratus.To investigate the composition difference in the ositions . Two RLRetazoans . The RLRstigated , which iRLRs can be attributed to multiple local tandem duplication events  that binds viral RNA and N-terminal CARDs to interact with the signal adaptor. We proceeded to explore the domain architecture of the RLRs. With the inclusion of the canonical vertebrate types (V-type), we classified metazoan RLRs into 11 major types based on their domain architecture  and one V2 type (LGP2). In the ancestral branch, Porifera (A. queenslandica), two A1-type RLRs with death as the N-terminal domain were annotated. Nine of the ten cnidarians were identified to possess two or three RLRs, and most (89%) cnidarian anthozoan RLRs were C (cnidarian-type), with only one CARD domain in the N-terminus. The types of RLRs that only occur in one species are classified as group X, which includes four species (X1–X4). Intriguingly, the domain architecture of RLRs showed the most striking diversity in lophotrochozoans, which contain the eleven divided RLR types. The N-termini of those eleven RLR types include: canonical V1 and V2 types, the A1 type of the death domain, the C type of the N-terminal CARD, the six L types (L1–L6) obtained from lophotrochozoans, and the X type. This observation may reflect a high level of domain grafting, resulting in significant expansion of RLRs in lophotrochozoans.Canonical itecture . Most   IG, DED,  SAM) in this cluster were annotated. In Cluster II, the RLRs with N-terminal IG and DED domains were found to be independently clustered, which suggested that the L2 (DED) and L4 (IG) types of RLRs were independent in lophotrochozoans. To determine the molecular evolutionary history of the oan RLRs . This trster II) . ClusterRLR gene family based on the intermediate helicase domain (DEXDc + HELICc domains)  . GeneralRLRs in lophotrochozoans, we conducted phylogenetic analysis using sequences of N-terminal domains of RLRs in lophotrochozoans, the ancient death domain in porifera, and the CARD domain in cnidaria -type RLRs, DED and IG clustered with the CARD1 branch, while SAM and CASc clustered with the CARD2 branch. These results indicate that the highly diverse and dynamic N-terminal domains in lophotrochozoan RLRs could have independently emerged from domain grafting. To clarify the evolutionary relationship of the diverse N-terminal domains of cnidaria . The twotic tree a, the evRLRs. Our hypothesis was tested by identifying sibling paralogs/orthologs with the highest sequence similarity based on the RIG-I_C-RD phylogenetic tree and comparing their exon–intron architecture  and calculated the tissue expression levels of RLR genes in ten evolutionary representative species , found two published databases from bivalves C. gigas and Scapharca broughtonii, and calculated their expression profiles (RLRs in C. gigas were significantly upregulated (Log2 (FC) > 1.5) during oyster herpes virus infection. Of these RLRs, CgiRLR6 encodes a lophotrochozoan-specific N-terminal IG domain. Similarly, S. broughtonii SbrRLR3 encoding only one CARD at the N-terminus was significantly upregulated (Log2 (FC) > 1.5) under virus challenge. These results suggest that lophotrochozoan RLRs with special domains may play an important role in antiviral immune recognition. We determined whether the duplicated lophotrochozoan profiles b. A totaRLRs must rapidly evolve and are thus subjected to positive selection pressures [RLRs with diverse N-terminal domains was driven by natural selection. Accordingly, a positive analysis was performed on lophotrochozoan RLRs with diverse N-terminal domains and gene expression levels enriched in the immune-related tissues mentioned above. We used the RIG-I_C-RD domain of RLRs for selection analysis, as this part is the virus recognition domain. As innate immune receptors are responsible for severe diseases, ressures . We deteRLRs of M. yessoensis, all of which were in Cluster I of the phylogenetic tree  superfamily. By mediating homotypic interactions within each domain subfamily, these proteins play important roles in the assembly and activation of apoptotic and inflammatory complexes [NLR (nucleotide oligomerization domain (NOD)-like receptor), recruited the apoptosis-related domains pyrin and baculovirus inhibitor repeats during evolution, leading to the control of the activation of inflammatory caspases in animals [RLRs with a CASc domain might be involved in apoptosis, inflammation, or pyroptosis. Molecules with IG-like domains are involved in a variety of immunological functions, including adaptive immune receptors, innate immune molecules, and accessory molecules [RLR is responsible for initial pathogen recognition, the N-terminal domains are required for downstream signaling [RLRs in lophotrochozoans. These new integrated non-canonical protein domains might play an unidentified role in innate immunity or host defense. In vertebrates, the N-terminal CARD domain of smission ,32,33,50C. gigas . Of noteomplexes ,52. The omplexes ,54. The omplexes . Previou animals ,57. We holecules . Previouolecules . One of olecules . Althougignaling ,30. NoveRLRs integrated with non-canonical N-terminal domains were distributed unevenly across the RLR phylogeny with the dominant clade in Cluster II, and most were species-specific. Most lophotrochozoan RLRs clustered with vertebrate RIG-I instead of MDA5/LDP2 and were divided into two clusters. The domain composition showed that the functions of the RLRs in Cluster II were more diverse than those in Cluster I. Previous studies have shown that in addition to RLRs [NLR and TLR, underwent massive species-specific expansions and domain shuffling in various lineages [RLRs (except CARD), the death, DED, and IG domains have emerged multiple times in different lineages, while the CASc and SAM domains have only been found in one species. Two separate domains of TLR, the TIR domain and the LRR domain, have been found to have a domain combination phenomenon. The combination of the P-TIR and P-LRR domains occurred in non-bilateria, while the early combination of the V-TIR and V-LRR domains for V-TLR occurred after the divergence of bilateria and non-bilateria [KIRs) is expressed on the surface of NK cells, which leads to the production of new KIRs [The phylogenetic results suggest that the lophotrochozoan  to RLRs , the othlineages . In the lineages . Studieslineages . In contilateria . Generalilateria ,62. In anew KIRs .RLRs. The structure of the RLR protein is relatively simple in ancestral metazoan porous animals and cnidarians. However, in lophotrochozoans, the domain arrangement of the RLR protein showed a complex and diverse pattern with the addition of the second CARD (CARD1) and non-canonical N-terminal domains. Deletion and incorporation of specific domains have become prevalent. Based on our results, the diversity of the N-terminal domains in lophotrochozoan RLRs should be attributed to domain shuffling after gene duplication. As the domains are often correlated with exon boundaries, exon shuffling is believed to be one of the major forces driving domain shuffling [KIR gene family. For KIR genes, the exon–intron structure correlates with the four main parts: the first three parts comprise an Ig domain, D0, D1, and D2, and the last part comprises the stem (S), transmembrane (TM), and cytoplasmic (CYT) domains [KIR being more favored by natural selection, another important reason for domain shuffling is that certain introns are hot spots for recombination [RLR gene, almost all specific domains were found to be fully encoded by only one exon   or from other databases  database . Multiple biotic stress data included PRJNA450478 (S. broughtonii after Ostreid herpesvirus-1 infection), and PRJNA146329 (C. gigas larvae infected with OsHV-1). Adult tissue data included PRJNA393252 ; PRJNA393252 (N. geniculatus); PRJNA286275 (L. anatina); PRJNA658966 (O. bimaculoides); PRJNA488641 (H. rufescens); PRJNA146329 (C. gigas); PRJNA578350 (M. coruscus); PRJNA185465 (C. farreri); PRJNA259405 (M. yessoensis); PRJNA608692 (E. foetida). To identify the atabases . TranscrRLR genes in 58 metazoan species (http://hmmer.org/download.html (accessed on 1 June 2021), was used to identify the RLR genes by screening the RIG-I_C-RD (PF11648) domain on the genomes of all species. Second, regions of each genome potentially harboring RLR genes were identified using TBLASTN  [http://blast.ncbi.nlm.nih.gov/, accessed on 4 March 2022) to further confirm this conclusion. Third, according to the structural characteristics of RLRs in a broad sense, we used the visualization software SMART  [RLR genes of each species containing an intermediate DEAD/DEAH box helicase domain and a RIG-I_C-RD domain. Finally, we corrected some RLR genes with suspected domain deletions or special domains using local GeneWise software  [RLR gene model studied.We used domain prediction and a sequence homology search of the  species . First, MD, USA)  with RLRer 2021)  to screeer 2021)  and tranRLRs identified by PFAM HMM were used to conduct the phylogenetic analyses in the present study. The protein sequences encoding the RIG-I_C-RD domain were aligned using the L-INS-I strategy in the local MAFFT v7.310  [https://itol.embl.de/, accessed on 1 November 2021). In addition, in order to explain the overall evolution of RLRs, the N-terminal domain coding sequences and intermediate helicase domain (DEXDc + HELICc domains) coding sequences were extracted to construct phylogenetic trees according to the same method.The highly conserved sequences of the RIG-I_C-RD domain of n, 2013) . Maximumn, 2013) , which cRLR gene family. However, the homologous gene pairs found by this method were often too limited, so we only used the method of phylogenetic analysis to identify RLR gene homologous pairs containing special lophotrochozoan RLRs.First, the local BLAST version2.9.0  was used for homologous gene pair identification by reciprocal BLAST searching on the retrieved datasets . Then, tTo determine whether paralogous or orthologous genes have diverged in exon–intron structure, we compared their genomic sequences. Two paralogs or orthologs were regarded as structurally divergent if they had different numbers of exons, or if they had the same number of exons but the lengths of at least one pair of homologous exons were different. To understand the underlying mechanisms of structural divergence, we generated pairwise alignments for each gene pair, using the corresponding mRNAs as guidance. Intraexonic insertion/deletion was deduced when an indel was found within the aligned homologous exons. Exon/intron gain/loss was inferred if an orphan exon/intron was the result of exon duplication, exon shuffling, exon scrambling, intron insertion, or intron deletion. Exonization/pseudoexonization was identified when the corresponding exonic and non-exonic sequences could be aligned with confidence .http://hannonlab.cshl.edu/fastx_toolkit/index.html, accessed on 1 November 2021) was used to process the raw reads to evaluate sequencing quality and remove low-quality reads , adaptor sequences, poly-N, and known non-coding RNAs (RLR genes (DEGs) were identified with the edgeR tool of the R programming language with the threshold value |log2FC| ≥ 1.5  and FDR ≤ 0.05. The FastxToolkit pipeline  with default parameters, and the resulting alignments were refined with trimAl version1.2 [Multiple sequence alignments of , Spain)   was used to locate and visualize the 3D structure of RLRs.PAML version4.9j   was usedRLR gene family in lophotrochozoans by investigating their domain architecture, phylogeny, exon–intron structure, expression profiles, and selection patterns. Our study revealed many previously unknown N-terminal domain fusions in lophotrochozoan RLRs, which might result in the diversity and specificity of the innate immune response. We traced the exon–intron structure of these non-canonical lophotrochozoan RLRs and found that the mechanism of exonization/pseudoexonization might drive the formation of these RLRs. Many non-canonical lophotrochozoan RLRs exhibit positive selection signals and immune-related expression patterns, indicating that non-canonical lophotrochozoan RLRs might have a selective advantage for organisms in the innate immune response. Overall, our findings suggest that the complex and unique domain arrangement of lophotrochozoan RLRs might result from rapid domain grafting, exon–intron structural divergence, expression diversification, and positive selection, which may have led to functionally distinct paralogs or orthologs in the innate immune response. Our research provides new insights into the molecular evolution of innate receptors in invertebrates in the absence of antibody-mediated adaptive immunity.In this study, we systematically described the evolutionary history of the


Escherichia coli in 25 selected Finnish piglet-producing and finishing herds that formed nine birth-to-slaughter production lines. Moreover, we studied associations between AMU and AMR in both herd types and throughout the production line. Treatment records were obtained from the national Sikava register for 1year, and AMU was quantified as mg/PCU (population correction unit) and TIs (treatment incidences). For phenotypic antimicrobial susceptibility testing, ten pen-level pooled feces samples (n=250) in each herd were collected from one room representing the oldest weaned piglets or the oldest finishing pigs. Majority of the medications (96.8%) was administered parenterally, and penicillin was the predominant antimicrobial in every herd. More different antimicrobial substances were used in piglet-producing than in finishing herds . As mg/PCU, sows had the highest AMU and suckling piglets had the highest TIs, whereas finishing pigs were the least treated age group. The proportion of susceptible indicator E. coli isolates of all studied isolates was 59.6%. Resistance was found most commonly against tetracycline, sulfamethoxazole, trimethoprim, and ampicillin, and multi-resistant (MR) isolates  were resistant to a maximum of four different antimicrobials. Quinolone resistance was rare, and no resistance against 3rd-generation cephalosporins, meropenem, azithromycin, colistin, gentamicin, or tigecycline was detected. The main associations between AMU and AMR were found at antimicrobial group level when use was compared with the presence of AMR phenotypes. The proportion of resistant isolates was not associated with AMU, and herd size was not associated with either AMU or AMR. We suggest that the use of narrow-spectrum beta-lactams as a primary treatment option and lack of wide application of oral group medications potentially favors a good resistance pattern in integrated pork production.In pigs, antimicrobial use (AMU) practices vary at different production phases between herds and between countries. Antimicrobial resistance (AMR) development is linked to AMU but recognized as a multi-factorial issue, and thus, any information increasing knowledge of AMU and AMR relationships is valuable. We described AMU and screened the carriage of different AMR phenotypes of indicator  In Finland, the prevalence of infectious pig diseases is generally low , and vetAttempts to harmonize the collection of both AMU  and AMR Escherichia coli bacterium is a widely used indicator organism due to its commensal nature and abundance in the intestines  sold their piglets at about 30kg body weight to 15 finishing herds (F1-F15), where the pigs were reared to about 110kg body weight. One of the piglet-producing herds (P10) bought weaned piglets weighing on average 7kg and reared them to about 30kg body weight.The monthly number of pigs present on each herd for the year 2018 was obtained from the Finnish Swine Registry system authorized by the Finnish Food Authority. Farmers are obliged to report their animal numbers, including suckling piglets, weaned piglets, sows, boars, young breeder pigs (gilts), and finishing pigs, to the system. In 2018, the median number of sows in P1-P9 was 896 , the total number of weaned piglets raised in P10 was 83,911, and the median number of finishing pigs farmed in F1-F15 was 13,320 .Treatment records of each herd for the year 2018 were collected from the Sikava register, where the herd owners saved their medicine use in electronic form. Data on antimicrobial treatments were extracted among all treatment records, and the AMU data included age group-specific information on the number of pigs treated, antimicrobial products used, doses, and administration routes . Age groups were suckling piglets, weaned piglets, finishing pigs, sows, boars, and young breeding pigs. Antimicrobial agents used  were grouped as follows: penicillin, beta-lactams other than penicillin, sulfa-trimethoprim, fluoroquinolones, tetracyclines, macrolides, lincosamides, and pleuromutilins.The total biomass of suckling and weaned piglets, finishing pigs, and young breeding pigs was obtained by summarizing the monthly numbers of each group multiplied by the standard weight of the animals. For sows and boars, the average monthly number of animals over 12months was calculated. The standard weights used for calculations were as follows: 4kg for suckling piglets, 12kg for weaned piglets, 50kg for finishing pigs, and 220kg for sows . The corFormula 1 Treatment incidence (TI) calculation according to The study herds were visited once between March and October 2018. For feces collection, one room housing the oldest weaned piglets in P1-P10 and one room housing the oldest finishing pigs in F1-F15 was selected. Ten evenly distributed pens were sampled (one pooled sample per pen) that in piglet-producing herds contained on average 18 weaned piglets and in finishing herds 6–15 pigs per pen. Sick pens were excluded. Altogether, 250 pooled samples were taken, and for one sample, three fresh feces piles were swabbed with one sterile cotton swab that was inserted into a culture medium tube . Samples were stored inside a cool box and transported to the laboratory, where the sample analysis started within a median of two (range 1–6) days.™ Orientation, Paris, France) and incubated at 37°C for 20–24h. A typical E. coli colony was picked from each plate  and streaked on trypticase soy agar plates  and incubated at 37°C for 20–24h. The colonies were confirmed as E. coli by Gram-staining, oxidase test, and API 20 E biochemical test assay . The isolates (n=250) were stored at −80°C until susceptibility testing.Samples were enriched in buffered peptone water (BPW)  and incubated at 37°C for 16–20h. A loopful (10μl) of enriched sample was cultured on selective media  was used for the antimicrobial susceptibility testing of indicator E. coli according to the manufacturer’s instructions. Phenotypic susceptibility was determined by using minimal inhibitory concentration values for the following 14 antimicrobials: ampicillin, azithromycin, cefotaxime, ceftazidime, chloramphenicol, ciprofloxacin, colistin, gentamicin, meropenem, nalidixic acid, sulfamethoxazole, tetracycline, tigecycline, and trimethoprim. For the quality control, E. coli ATCC 25922 was used as a reference strain. The susceptibility results were categorized as either wild type (with reference to susceptible) or non-wild type (with reference to resistant) based on epidemiological cutoff values (ECOFFs) defined by the European Committee on Antimicrobial Susceptibility Testing  was used for statistical processing of the data, and herd was used as the experimental unit in all statistical analyses. Data normality was visualized with histograms. Quantification of AMU was done for the year 2018, and antimicrobial susceptibility testing for each herd was conducted once within that year. We expected, however, that the AMU of the study herds is steady throughout the year, thus justifying the comparison between longitudinal AMU and cross-sectional AMR parameters.n=10). Herd AMR was calculated by dividing the number of different antimicrobial agents included in the susceptibility testing panel to which studied isolates were resistant with the total number of antimicrobial agents (n=14). The percentage of phenotypes that were the same both in piglet-producing and finishing herds out of all phenotypes detected in piglet-producing herds was calculated to investigate phenotypic resistance at production line level. Three most common phenotypes present in both herd types within a production line  of the herd was tested with Spearman correlation. Similarly, we tested whether the average number of sows and the total number of suckling and weaned piglets were associated with AMU (mg/PCU) of the respective age groups.For the outcomes “proportion of resistant isolates in the study herds” and “herd AMR,” linear regression models were built for piglet-producing herds and mixed effect regression models for finishing herds. Models for piglet-producing herds contained total AMU per herd and herd size as fixed effects. For finishing herds, production line was included as a random intercept in both models. In piglet-producing herds, the associations between AMU (mg/PCU) for separate age groups, the proportion of resistant isolates, and herd AMR were investigated with Spearman correlations. For the same outcomes, we examined whether the use of separate antimicrobial groups as milligrams and TIs of separate age groups was associated with AMR by using Spearman correlations.n=15) in a herd,” Spearman correlation was used. The following explanatory variables were included in comparisons: total AMU for both herd types and total use of separate antimicrobial groups as milligrams, use by age group , and TIs of different age groups separated by antimicrobial group. Total AMU and number of different antimicrobial agents used in piglet-producing herds were compared with herd AMR and with the presence of different resistance phenotypes (n=14) in finishing herds by using Spearman correlation.To investigate whether total AMU for both herd types and separately for suckling piglets, weaned piglets, and sows (mg/PCU) were associated with the number of different AMR phenotypes, Spearman correlation was used. Additionally, total use of separate antimicrobial groups as milligrams and TIs separately for all age groups was compared with the number of different resistance phenotypes by using Spearman correlation. The association between the number of different antimicrobial agents used in herds and the number of different resistance phenotypes by using categorical variables was examined with chi-square test. For the binary outcome “occurrence of phenotype X  compared to finishing herds  . Altogether 190,212 pigs had been treated with antimicrobials in 1year: 96.8% parenterally and 3.2% orally Considerable variation existed in AMU (mg/PCU) between herds and age groups . The useE. coli isolates studied were fully susceptible. Of 101 resistant E. coli isolates, 53.5% were R and 46.5% MR. Overall, the average proportion of isolates resistant to at least one antimicrobial agent was 40.4% (SD 15.1%), being 43.0% (SD 17.7%) in piglet-producing herds and 38.7% (SD 13.6%) in finishing herds. Resistance was most common against tetracycline (29.6%), sulfamethoxazole (22.0%), trimethoprim (21.2%), and ampicillin (15.2%) (Altogether 59.6% of 250 indicator  (15.2%) . A few oOverall, the resistance patterns of the studied isolates comprised 15 different AMR phenotypes .In an average of 60.6% (SD 28.8%) of the production lines, the resistance phenotypes of indicator p<0.01).Total AMU, AMU at age group level , or number of different antimicrobials used were not associated with AMR outcomes, including proportion of resistant isolates in the study herds, herd AMR, number of different resistance phenotypes, or the presence of single AMR phenotypes. In finishing herds, total AMU was negatively associated with herd AMR  and the presence of the SMX-TMP-TET-AMP phenotype  in finishing herds. Moreover, total AMU in piglet-producing herds was negatively associated with the presence of TET phenotype in both herd types .Total AMU or number of different antimicrobial agents used in the piglet-producing herds (1–6 per herd) was not associated with the herd AMR in finishing herds or with the presence of any particular resistance phenotype. There was a negative relationship between the number of different antimicrobial agents used in piglet-producing herds and the occurrence of the same phenotypes in the finishing herds  of all pigs treated. Even though AMU of the individual study herds was rather variable, narrow-spectrum beta-lactams, especially penicillin, was used predominantly in each herd. This implies that both the administration route and the selection of antimicrobials for treatment followed national  and Eurovia feed, whereas no antimicrobials were given via drinking water. Contrary to our results, prophylactic oral group treatments are reported to be common, especially during the post-weaning period in many European countries  in either piglet-producing or finishing herds. Similarly, herd size was not associated with the proportion of resistant isolates. Likewise, The present study included a convenience sample of herds from selected geographical areas and with integrated production. Although the Sikava register has relatively detailed data on AMU, it may contain some erroneous treatment records. In addition, general practice among farmers is to maintain their medical bookkeeping first on paper, then transferring it to the register in electronic form. To investigate the associations between AMU and AMR, more detailed AMU quantification methods are needed. Currently, there is no harmonized and precise AMU indicator at herd level , especiaE. coli bacteria studied were fully susceptible, and overall, the resistance pattern followed a trend similar to those reported both nationally and in Europe over several years. Resistance was most common to the antimicrobials generally used in pigs. A novel feature in the present study is that we evaluated the herd-level phenotypic AMR pattern throughout the pork production lines and included a comparison between production lines. Our results did not reveal a positive association between herd-level AMU and AMR. The linkage between AMU and AMR is complicated, and thus, any information shedding light on AMR development is valuable. Taken together, it is important to standardize the unit of AMU measurements and to assess the impact of AMU selection pressure on AMR development in more detail.Our findings indicate that primarily narrow-spectrum beta-lactam antimicrobials were administered parenterally to single pigs. A surprising finding was higher AMU in sows than in other age groups. Almost half of the indicator The original contributions presented in the study are included in the article/The animal study was reviewed and approved by Southern Finland Regional State Administrative Agency (ESAVI/16950/2018). Written informed consent was obtained from the owners for the participation of their animals in this study.VS, SN, AH, TT, and MH participated in the planning of the study. VS, TT, and MH conducted the herd visits. VS and OH performed the statistical analyses. All authors participated in the writing process and approved the final manuscript.This research was funded by the Finnish Centre for Economic Development, Transport, and the Environment (project number 58451) as part of the Rural Development Program for Mainland Finland 2014–2020 and by the Walter Ehrström Foundation.The Finnish slaughterhouse company Atria Finland Plc participated in the study by recruiting the study herds but did not influence the interpretation or reporting of results. One of the authors (TT) is employed by the slaughterhouse company.The remaining 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.


Focus group discussions (n = 12) were conducted and thematic analysis applied using an inductive, semantic, contextualist approach. Five themes were identified: (1) Contribution to food security; (2) Better sensory and baking properties than local flour; (3) Perceived health benefits; (4) Willingness to pay for the flour; and (5) Importance of trusted promoters/suppliers. Although the participants were blind to whether they had received control or biofortified flour, referred to collectively as “study flour”, the results indicated that the study flour performed well in terms of its taste and bread making qualities, with no adverse reports from participants in either arm of the BIZIFED2 RCT. Participants suggested that they would buy the biofortified wheat if this was available at a fair price due to perceived health benefits, reporting positive sensory characteristics and cooking attributes when compared to the flour available in the local markets. Overall, there was a positive reception of the programme and flour among the participants, and members of the community hoped for its continuation and expansion.Zinc-biofortified flour may be a cost-effective approach to improve zinc status of populations in low-resource settings. The success of biofortification programmes is subject to acceptability and uptake by consumers. This study explored community leaders’ and community members’ ( Zinc deficiency is estimated to affect approximately 17% of the world’s population , with muBiofortification is the process by which the nutritional quality of food crops is improved through genetic engineering, conventional breeding or pre-harvest agronomic practices . These pA “high-Zn” wheat variety Zincol-2016) was developed by Pakistan’s National Agriculture Research System (NARS) using traditional breeding techniques with support from HarvestPlus and was released by the Pakistan Agriculture Research Council (PARC) in 2016. This variety of wheat, when grown with zinc fertilisers, contains more zinc per kilogram of grain compared with traditional varieties . The Bio was deveThis study is part of the BiZiFED2 project (Biofortification with Zinc and Iron for Eliminating Deficiency), funded by the United Kingdom Research and Innovation (UKRI) Global Challenge Research Fund (GCRF), which started in April 2019 and will conclude in March 2022. BiZiFED2 included a cluster-randomised, double-blind, controlled trial designed to test the effectiveness of zinc biofortified wheat flour for improving zinc status of adolescent girls and children in rural Pakistan . The stun = 250, provided with flour milled from the biofortified strain of wheat (Zincol-2016)), or the control arm BiZiFED2 RCT . An earlier randomised controlled efficacy study conducted by the same research group reported that Zincol-2016 wheat, grown under optimal conditions of zinc fertiliser application specifically for this study, was able to achieve a mean zinc concentration of 49.3 mg/kg compared to 22.2 mg/kg in Galaxy-2013 grain , no one can afford meat, so it is just the flour we consume it and we consume it eagerly.”(A woman)However, some female participants perceived the distribution to be insufficient to satisfy the needs of their entire family:“They give me 3 bags which are not enough for a month for me, so I buy 2 small bags, we are 12 to 13 people (family members) 3 bags are not enough for a month for me. We have to eat other flour because this flour isn’t sufficient for me.”(A woman)“We are very happy for receiving the flour. We pay back the money that we save by receiving this flour to those we had borrowed from. So, this scheme saves money for us and keep our oven fine (chuckling), our stomach linings are good with it. We can’t afford fruit due to the poverty. All these people are poor, and we are very grateful for receiving this”.(A woman)The provision of free flour had a positive impact on household finances and enabled some participants to use the money they saved to purchase other foods or for alternate purposes:Some participants highlighted the importance of good local storage facilities in order to ensure the continuous distribution of biofortified flour. Disruptions in flour availability meant that individuals had to rely on borrowing additional supplies from neighbours or family members.“There was one issue that it wouldn’t cover the whole period of month uninterruptedly when not delivered on time [so they would have to use other flour during that gap]. That issue was there in the initial months but when they arranged this store here and the goods came here then there hasn’t been any shortage until this month, in this month there is a shortage, our flour is finished and we (borrow) it from one and other home.”(A jirga member)Any negative impact on food security due to COVID-19 pandemic-related restrictions appeared to be alleviated by participation in the study.“The conditions deteriorated very much during the Corona, everything was affected yeah, but we were happy that at least we have been receiving this one relief, our food was good.” (A jirga member). Another Jirga member said, “There was a lot of difficulty, there was shortage for some time, the vehicles were banned absolutely, and if this flour were not available people would have had a big problem.”(A jirga member)There was a perception among participants that the project logistics team efficiently managed the distribution during the pandemic.“We wouldn’t go as a big crowd, they would give us a schedule telling us this is when your turn is, so there would be no crowd created. Because of that the process was a bit slow but other than that we would receive a call on time to let us know that your share has arrived. So, it was handled well during the pandemic (A jirga member). No we have not, we received it regularly on time.”(Another jirga member)The participants were satisfied with the flour they received, commenting that it was cleaner, had better fermentation properties and produced better bread than the flour that they bought in their local market.“When the bread of the bazaar [flour] cools down for a while then it turns into something like a rubber, it becomes firmer. But this flour becomes like (our very own) wheat. Its colour is also good, white colour. This flour is much better flour than the bazaar one; our females tell us this is very easy for them in fermenting, they say wetting it is very easy for them (A man). sir, there is absolutely difference between this one and the bazaar one.”(Another man)Some of them expressed that there was consistency in the quality of the flour received, whereas the flour available in the market would sometimes be mixed with other things or that they did not believe that they received what they had paid for.“There is a big difference; the big difference is that there is no idea what is getting mixed into the other flour. The cold (old) bread is also mixed into it and this is something I saw with my own eyes in a flour mill.”(A man)As participants of the RCT all FGD participants consumed either control or biofortified flour. Despite being blinded to which arm of the trial they had been allocated to, most participants perceived the study flour to have health benefits, stating that since they started consuming the flour provided their families had fewer stomach problems, fewer respiratory infections and more energy“Our throats used to have a problem all the time, all the time but since we have started this flour, we got rid of that (throat problem). When we were eating the common flour, we had to take stomach tablets in the morning but since we have started this flour, we don’t have any stomach problem, our stomachs are fine. Since we have started, we didn’t have any throat issue and even we didn’t have a flu. All of us and our children became stronger. There isn’t any (problem) with it, and we want this project to run further because it is beneficial, and it has benefited us very much.”(A man)Both male and female participants expressed concerns that their health may deteriorate when the programme came to an end and they stop receiving the flour.“We hadn’t eaten flour like this before. Stomach and abdomen and so on also haven’t got any problem with it. But the issue is that they say that after one and a half month or two months, this is going to finish so it means we will go back to the situation we had been in before. So, our request is that whoever is behind this decision they would listen to us and so they keep this continuing for some more time.”(A man)“We are in good health with this, but we don’t know what to do as now our health is going to deteriorate.”(A woman)The perceived health benefit was one of the reasons participants gave for their willingness to buy biofortified flour once the project had ended.“I will certainly buy it because we have used it and we know it. It helps our health and brings energy therefore we will definitely be buying this flour. (A man) It is absolutely like that, I mean, if we stopped receiving the flour, and it were available at the market and if it is in a shop in the market then we will be buying it as we know the benefits of it.”(Another man)An important factor guiding the community member’s decision to buy a particular type of flour related to its price. The participants were willing to pay a slightly higher price for biofortified flour due to its quality and perceived health benefits.“We are poor people… so the price should be reduced for us, but when you bear in mind it’s better quality then if it is 200 (USD 1.14) or 100 [rupees] (USD 0.57) more expensive, it will be fine. It is good.”(A woman)“The reason we will be buying this one is… even if this is ten or eight rupees (USD 0.057) more expensive then so be it as all praise be to Allah we are protected from illness and problems that children would have... We are labourers and we would miss our labour work [for taking our children to a doctor]... So we do not have to bother about those problems anymore and that’s why we are using this flour, so if this flour comes out in the market by the grace of the holy Allah, then we will be using it.”(A man)Some of the participants said that they will continue buying biofortified flour as a consequence of family demand.“In terms of baking, we ask our women about (and they say) it doesn’t get over yeasted, and they say this (flour) is good whether it is baked in a saucepan or in an oven it comes up with the same beautiful bread. So, our family will tell us to bring this one and we will certainly bring it. When we do our labour work then we will certainly buy this flour bag.”(A jirga member)The participants hoped for the flour to continue to be available after the programme ended, and some expressed their willingness to grow the biofortified variety on their land.“We request the government of Pakistan through [NGO working in the community] to make this flour available to the whole of Pakistan, particularly to [Area] and more particularly to the [District] (multiple voices) yes.”(Men)“We are ready to grow it in the fields we have and our own land, or the lands that we have leased.”(A jirga member)An important aspect relevant to future biofortification scale up activities related to the importance of trust between community members and the providers of the flour. This trust was also important in the context of the intervention trial where participants were asked to provide blood samples to monitor health status. Participants reported that some community members who had not participated in the trial were sceptical about the programme and had criticised their neighbours for agreeing to take part in the study.“They do say and ask questions that ‘you are giving blood which affects your children later on, and they [the researchers] want indecency in the society, this is from the foreign countries’, so they say this sort of thing.”(A man)However, these participants believed that such comments were linked to feelings of jealously, adding that the comments did not concern them since they trusted the NGO and the individuals running the programme and perceived it to be of benefit to them.“As far as people are concerned, those who don’t receive it and they are not in this then they do make dramas about this in the neighbourhood and vicinity [saying] this is this thing from the English people, this is that thing of the English people, but we don’t believe it because the flour benefit us.”(A man)“We know [member of staff] very well … I mean we know all of them so, God willing, they will not do anything wrong to exploit us and God willing they will work for our benefit as long as this programme runs.”(A man)“Everyone has their own opinion, yeah, and they behave according to that. But this flour didn’t give us any harm, on the contrary it benefited us. Loss and gain is in the hands of Allah but human does something with good intention. They [NGO working with the community] are working with good intention. Allah will give them a reward.”(A man)Some of those who had expressed some initial mistrust had changed their perception during the course of the programme, expressing their wish to be included in the study.“They say good things and also bad things as well, however now they don’t say bad things, they say good things. (A man) Now all of them have become assured about this thing and they say if there is a chance for them to be admitted into.”(Another man)This qualitative study explored the experiences and attitudes towards the study flour among households and Jirga from communities participating in the BiZiFED2 RCT . UnderstParticipants reported that they would be happy to buy biofortified wheat if it were available in the local market. Consistent with previous studies ,17, partUnsurprisingly, participants also emphasised the importance of affordability and price. Participants who had access to land were eager to receive seed for the biofortified wheat variety so they could cultivate it themselves. Studies to assess biofortification costs are required, including additional production costs , marketing and social behaviour change programmes and monitoring costs. In turn, affordability needs to be assessed including for the poorest households, and this can inform how to make scale-up sustainable.Overall, there was good acceptance of the BiZiFED2 RCT within the community that participated in the trial. This acceptance may be attributed in-part to the relationship of support and trust that the Jirga and the collaborating NGO have established with over 20 years of working in this area. Jirga members, teachers, medical doctors and religious leaders are seen as trusted members of the community and during the preparation for the trial, Jirga members expressed their feeling that it was part of their role to raise awareness about the study and provide reassurance about the project to the community . These fA major strength of this study was its in-depth approach, generating rich, qualitative data from a relatively large number of participants including men, women and Jirga in the community. There were, however, some limitations to our study. BiZiFED2 was a blinded trial and the FGDs included individuals from control and intervention households. Therefore, participants were only able to report on the attributes of ‘study flour’ and there was no way to distinguish differences between the responses of consumers of control and biofortified flour. Furthermore, participant responses may have been influenced by the receipt of free flour which may have led them to exaggerate positive aspects of the programme and the study flour. Issues of consumer acceptability outside a research setting should be investigated further. Likewise, participants were drawn from households engaged in the BiZiFED2 RCT and Jirga members from respective communities, and the opinion of these participants may not be representative of the wider community, including those who were not selected to take part in BiZiFED2 RCT, or those who declined to participate. Nonetheless, the findings still present useful information and learnings to inform future research studies and programmes that seek to scale-up biofortified wheat in Pakistan.In this study, we assessed attitudes towards the flour provided in a biofortification trial from a consumer perspective. Further research should explore the perspectives of producers and potential vendors, including their current practices and beliefs, attitudes, potential challenges and motivators towards producing and selling biofortified wheat.Our study found that there was good acceptability amongst the community towards the flour provided in the BIZIFED2 RCT, with participants reporting perceived health benefits and good cooking/sensory qualities. Participants felt that the flour was of better quality in comparison with local wheat flour. The distribution of flour was also welcomed and perceived both as a source of food security and economic support and in this low-resource setting. Our findings may be used to inform future research and scale-up programmes for biofortified wheat in Pakistan.


In vitro protein studies have identified assembly kinetics and the structural motifs involved in binding of adjacent LN domains. The physiological importance of these interactions has been identified through the study of pathogenic point mutations in LN domains that lead to syndromic disorders presenting with phenotypes dependent on which laminin gene is mutated. Genotype-phenotype comparison between knockout and LN domain missense mutations of the same laminin allows inferences to be drawn about the roles of laminin network assembly in terms of tissue function. In this review, we will discuss these comparisons in terms of laminin disorders, and the therapeutic options that understanding these processes have allowed. We will also discuss recent findings of non-laminin mediators of laminin network assembly and their implications in terms of basement membrane structure and function.The laminins (LM) are a family of basement membranes glycoproteins with essential structural roles in supporting epithelia, endothelia, nerves and muscle adhesion, and signaling roles in regulating cell migration, proliferation, stem cell maintenance and differentiation. Laminins are obligate heterotrimers comprised of α, β and γ chains that assemble intracellularly. However, extracellularly these heterotrimers then assemble into higher-order networks  Most BMeceptors , 2009. ALaminins are an obligatory feature of every BM. Each LM is an αβγ heterotrimer comprised of one of five α chains (encoded by LAMA1-5), one of four α chains (LAMB1-4) and one of three γ (LAMC1-3) chains . Use of The LM family has arisen by gene duplication and rearrangement, and common structural motifs are shared between members . ArchetyLaminin N-terminal domain are 252–264 amino acid globular domains found not only in LMs but also other ECM proteins including netrins. 14% of all residues in LN domains are conserved with strict conservation of six key cysteines. There is 72% homology between LMα1 and LMα2, 77% between LMα3B and LMα5, 72% between LMβ1 and LMβ2, and 64% between LMγ1 and LMγ3 LN domains. Lowest conservation is between LMβ3 and the β1 and β2 LN domains  .In vitro analyses have further shown that the αβγ ternary node assembly involves rapid but unstable formation of βγ pairs that are then stabilized through integration of an αLN domain , caused by mutations to LAMA2 (LMα2) . This afIn LMα2 knockout conditions, MDC1A presents with disabilities of the proximal and distal limb muscles, with patients unable to walk more than a few steps unaided . Weaknes1  has been associated with late onset Alzheimer’s disease , and dysAlthough the standard gene and protein therapy toolbox are available to treat LN domain disorders, the large size of LM genes and associated challenges of producing and delivering recombinant therapy-grade LM protein presents challenges. However, promising results have been obtained recently from delivering the 800 kDa LM521 to the blood stream of LAMB2-null mice which rescues some aspects of Pierson syndrome. The delivered LM521 accumulated in the glomerular basement membrane in the correct orientation and led to reduced expression of the podocyte injury markers, and delayed the onset of proteinuria. However, the exogenous LM521 did not migrate to the podocytes nor fully restore the glomerular filtration barrier. Smaller, or hybrid proteins, may be a solution to overcome these challenges . For somvia the nidogen region and provide the missing αLN domain needed to allow LM411 polymerization (A particularly innovative solution exploiting the knowledge gained from studying LM polymerization and counteracting the inherent LM size problems is using protein chimeras to act as linkers . Three srization . The minrization . Where pComparison between knockout and missense mutation associated phenotypes in LM genes has provided valuable information to identify which LMs are essential for individual tissues, but also which domains are involved. Rather than a binary outcome caused by ability or inability to polymerize, we see system-wide differences highlighting the multifaceted roles of LN domains. The variety of pathologies arising from mutations within a stretch of ∼250 amino acids illustrate the importance of LN domains to tissue function.LS, CS, and KH wrote and edited the manuscript. All authors read and approved the final version of the manuscript for publication.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.


Herein, we present an easy-to-use alternative that involves the combination of a new, methylated building block  with vacuum drying. Condensation of Me3TFB with 1,4-phenylenediamine (PA) or benzidine (BD) resulted in imine-linked 2D COFs (Me3TFB-PA and Me3TFB-BD) with higher degrees of crystallinity and higher BET surface areas compared to their non-methylated counterparts (TFB-PA and TFB-BD). This was rationalized by density functional theory computations. Additionally, the methylated COFs are less prone to pore collapse when subjected to vacuum drying and their BET surface area was found to remain stable for at least four weeks. Within the context of their applicability as sensors, we also studied the influence of hydrochloric acid vapour on the optical and structural properties of all COFs. Upon acid exposure their colour and absorbance spectra changed, making them indeed suitable for acid detection. Infrared spectroscopy revealed that the colour change is likely attributed to the cleavage of imine bonds, which are only partially restored after ammonia exposure. While this limits their application as reusable sensors, our work presents a facile method to increase the robustness of commonly known COFs.Covalent Organic Frameworks (COFs) are thermally and chemically stable, nanoporous materials with high surface areas, making them interesting for a large variety of applications including energy storage, gas separation, catalysis and chemical sensing. However, pore blocking and pore collapse may limit their performance. Reducing the capillary forces by using solvents with low surface tension, like supercritical CO Methylation of aldehyde nodes in Covalent Organic Frameworks leads to enhanced BET surface areas and reduced pore collapse compared to their non-methylated counterparts, which has been rationalized by DFT computations. This enables error correction, leading to crystalline frameworks with a long-range order. The symmetry of the building blocks is determining the crystal structure. In the case of 2D COFs, the third dimension is formed by π–π stacking. The layers usually stack in an eclipsed structure leading to channels in the framework, which results in high surface areas. These high surface areas are of interest for, amongst others, energy storage, gas separation, catalysis, and chemical sensing.3,6–8 The variability in organic building blocks in terms of functional groups or symmetry allow a high control over the COF properties and thus leads to tailor-made materials. However, the boronic esters used to make the first COFs were susceptible to hydrolysis. This has been improved by using imine-linked COFs or even more stable β-ketoenamine COFs. The latter first forms imine bonds, which then tautomerise irreversibly into the β-ketoenamine linkage. The irreversible tautomerisation results in a lower crystallinity and surface area compared to their imine counterparts.2 The chemical stability of COFs does not only depend on the type of linking chemistry, but can also be affected by the nature of the core of the organic building blocks. Ma et al.9 reported two highly porous 3D COFs of terephthalaldehyde or 4,4′-biphenyldicarboxaldehyde and a tetrahedral amine with isopropyl groups in ortho position. Isopropyl groups are hydrophobic and their steric demand protects the imine groups from hydrolysis. The COFs are reported to be stable in acidic and strong alkaline solutions without a decrease in surface area and crystallinity. Such stability was also achieved by methoxy substitution of the aldehyde node.10 In another study, Wang et al.11 reported enhanced BET surface areas for ortho methoxy-substituted aldehyde nodes and meta methyl-substituted amine linkers.Covalent Organic Frameworks (COFs) are a class of nanoporous materials first discovered in 2005 by Yaghi and co-workers.12 Capillary forces increase with decreasing pore size and COFs with typical pore sizes around a few nanometres are therefore subjected to strong capillary forces. To overcome pore collapse, milder activation methods such as supercritical CO2 drying12 or washing with ultra-low surface tension solvents prior to drying13 were suggested to keep the framework intact upon activation. Feriante et al.12 and Zhu et al.13 both reported methoxy-functionalised, imine-based COFs, which were less prone to pore collapse compared to the non-methoxylated equivalent.COF stability is not only about the chemical stability of the functional groups involved, but also about the stability of the physical properties such as pore size, pore shape and surface area. It has been identified that the conditions for work-up and COF activation are crucial to get accessible pores and high surface areas. After synthesis, COFs are most commonly washed with several solvents to remove impurities and water from the pores prior to drying. Vacuum drying has often been used, but was identified to induce partial pore collapse due to capillary forces.In the current paper, we introduce methyl groups in the synthesis of imine-based COFs as alternative to the bulky isopropyl and the smaller methoxy groups. Depending on the COF application, the use of this smaller – but still hydrophobic – group can be advantageous as it has less impact on the pore size.3TFB) as building block for two novel COFs, namely Me3TFB-PA and Me3TFB-BD, which are compared with two reference COFs TFB-PA6 and TFB-BD.14,15 The methylation significantly increases the BET surface area by a factor of 1.8 and 1.4, respectively. The physical stability of the framework towards pore collapse during vacuum activation also profits from the methylation. This work shows a facile approach in stabilizing COFs by methylating the aldehyde. The novel COFs maintain their high surface area on the bench for at least four weeks. Finally, the effect of acid vapour on the optical and structural properties of the COFs was studied.Herein, we present 2,4,6-trimethylbenzene-1,3,5-tricarb-aldehyde (Me3TFB) were condensed with two different amines, namely 1,4-phenylenediamine (PA) and benzidine (BD) (3TFB-PA and Me3TFB-BD) and their non-methylated equivalents (TFB-PA and TFB-BD). Each COF has been synthesised and characterised thrice. We selected this set of COFs, because TFB-PA and TFB-BD have been widely studied in literature.6,14–16 The synthesis was carried out at 70 °C and atmospheric pressure with acetic acid as catalyst and water to enhance dynamic covalent chemistry in a mixture of mesitylene : 1,4-dioxane 1 : 4 v/v for three days.17 A washing protocol from Vitaku et al. has been used for the work-up.18 Afterwards, half of the obtained powder was dried overnight in a vacuum-oven at 120 °C and the other half at 120 °C in a regular, air-ventilated oven before the COFs were characterised. The characterisation for the reference COFs can be found in the ESI  and 2,4,6-trimethyl-benzene-1,3,5-tricarbaldehyde (Meine (BD)  to yield3TFB-PA shows a characteristic imine stretch at 1623 cm−1, demonstrating a successful reaction. A weak band at 1693 cm−1 indicates low fractions of aldehyde bonds. This IR characteristic has also been reported by others and may origin from unreacted side-groups at the outside of the 2D polymeric sheet.12,13,19FT-IR spectroscopy on the novel COFs confirmed the formation of imine bonds . Me3TFB-13C cross-polarization magic angle spinning solid-state NMR (13C CPMAS ssNMR)   as the s3TFB-BD COF with an imine band at 1627 cm−1 and a small carbonyl stretch at 1693 cm−1  was performed on all samples to determine the crystallinity of the materials. The obtained spectra were compared to the simulated diffraction patterns of optimized crystal structures obtained from density-functional theory (DFT) calculations  was calculated using the Rouquerol criteria24 to apply the BET theory to microporous materials. For all samples of the same COF, the same pressure range was used for the linear regression. Afterwards, the obtained BET surface areas were averaged. For Me3TFB-PA SBET was found to be 1877 ± 20 m2 g−1. To determine the pore size distribution, HS-2D-NLDFT analysis was carried out using the isotherm data and a model with cylindrical micropores for carbon surfaces with N2. Me3TFB-PA COF shows a narrow pore size distribution with a clear maximum at 1.9 nm . The step in the isotherm at p/p0 = 0.1 indicates capillary condensation. The lack of hysteresis is in line with the cylindrical shape of the pores.22 The pore volume at p/p0 = 0.95 and BET surface areas were measured to be 1.24 ± 0.03 cm3 g−1 and 2115 ± 50 m2 g−1, respectively, while the pore size distribution has a narrow maximum at 2.7 nm  after vacuum activation , while both activation processes are equally good for Me3TFB-BD (retained SBET 97 ± 15%). In other words, the BET surface areas increase by a factor of 2.8 and 1.4 for Me3TFB-PA and Me3TFB-BD, respectively. The methylated COFs are hence less prone to pore collapse.To study whether the addition of methyl groups also results in a more stable framework, we studied the pore collapse by re-measuring the BET surface area of all COFs after vacuum activation . To correct for the difference in BET surface area due to different pore sizes, the retained BET surface area has been calculated as the ratio of the vacuum-activated tivation . The ret2 g−1 in the first four weeks, which means only 11% of the original BET surface area are accessible after one month. Therefore, we investigated the air stability of the other three COFs as well, by leaving the oven-activated samples for four weeks in an open vial on the bench, before determining the BET surface area again. These three COFs did not show such a large drop in the SBET values. Instead, on average a slight increase is observed (up to 8% for Me3TFB-BD), although the differences are within the standard deviation. In any case, as no decrease is observed, we conclude that TFB-BD, Me3TFB-PA, and Me3TFB-BD are stable for the reported conditions. Again, this shows that the introduction of methyl groups stabilizes the phenylenediamine COF.In addition, we noticed that the BET surface area of TFB-PA decreases already drastically to 113 ± 53 met al. observed a solvatochromic effect, which has been visible in diffuse reflectance spectroscopy, before employing the pyrene-containing COF for the development of a humidity sensor.8 Several studies report on COF-based chemosensors for the detection of gaseous hydrochloric acid,25,26 and a series of aqueous Brønsted acids.26,27 Recently, Yang et al. have studied the effect of imine-protonation on the optical properties of three imine-linked COFs. It was found that all COFs showed a bathochromic shift after acid exposure due to the protonation of the imine bond.28 Based on both findings, we looked into the absorption spectra of Me3TFB-PA and Me3TFB-BD and their response towards hydrochloric acid vapour. To this end, samples of the new COFs were pressed as a thin film between two glass plates. Absorption spectra were collected before treatment with acid by measuring the samples in an integrating sphere and collecting diffuse reflectance and diffuse transmission to calculate the absorbance. Both absorbance spectra have an inverse S-curve shape, start absorbing between 400–450 nm, and have absorption edges around 378 nm for Me3TFB-PA and 385 nm for Me3TFB-BD (3TFB-PA and 419 nm for Me3TFB-BD COF).The optical properties of some COFs are reported to respond to changes in acidity and solvent, enabling them to be used as chemical sensors. In more detail, Ascherl e3TFB-BD . To facie3TFB-BD , which wAs the two-plate configuration does not allow vapour exposure experiments, a new sample was taken from the same synthetic COF batch, which was subjected to hydrochloric acid vapour for 30 minutes. Within minutes, a colour change  was observed by the naked eye , which w3TFB-PA COF, although the first derivative indicates a wider range in which the absorbance changes and the absorption edge is red shifted with respect to the acid-exposed sample. For Me3TFB-BD COF, the spectrum also shifts back to higher wavelength, but even wider. The first derivative indicates two small minima with shifted positions that do not overlap with the original minimum, so the acid has an impact on the COF structure. The absorbance spectra for the non-methylated COFs can be found in the ESI  and 1685 cm−1 (carbonyl stretching vibration) are visible, indicating a partial recovery of the imine bonds.We speculate that the acid protonates the nitrogen atom . In a se3TFB-PA and Me3TFB-BD. They have been compared with non-methylated reference COFs regarding their BET surface area, pore collapse and their applicability as optical acid sensors. With a facile synthetic approach, which does not require any specialized equipment, we found that methylation of the aldehyde node results in enhanced BET surface areas up to ca. 2000 m2 g−1 and high degrees of crystallinity compared to their non-methylated equivalents. The obtained BET surface areas are comparable to the ones achieved by supercritical CO2 activation. The higher surface areas and crystallinity are in line with DFT-calculated reaction rates. With respect to TFB, Me3TFB loses symmetry and its full conjugation due to the ortho-substituted methyl groups. This leads to a smaller rotation barrier towards the favoured C3 conformation for crystallisation and therefore to higher reaction rates. Additionally, the novel methylated COFs are less prone to pore collapse during vacuum activation and the BET surface area remains stable within at least four weeks.In conclusion, a new methyl-substituted aldehyde building block has been used for COF synthesis, resulting in two novel frameworks, namely Meortho-substituted methyl groups of the amine building block and systematically increasing the number of methyl groups on the resulting COF properties are currently under investigation.The COFs can be used as an optical sensor to detect hydrochloric acid based on a change in colour. The sensing mechanism reveals that the colour change is likely attributed to the cleavage of the imine bond, which is partially restored after exposure to ammonia vapour. Even though this limits their applicability as a reusable sensor for hydrochloric acid, our approach enables the facile synthesis of high surface area COFs that are more robust compared to commonly known COFs. The effect of L.C.P.M.d.S. and E.D. conceived the project. E.D. and M.L. designed and performed the experiments. G.L. performed the modelling. E.D., M.L. and L.C.P.M.d.S. analysed all data. L.C.P.M.d.S. guided the project. E.D. wrote the first draft, and all authors gave input.There are no conflicts to declare.NR-013-D1NR05911D-s001NR-013-D1NR05911D-s002NR-013-D1NR05911D-s003NR-013-D1NR05911D-s004NR-013-D1NR05911D-s005