Agroforestry systems can potentially increase tree diversity within agricultural landscapes, but to date, there is little understanding of the patterns of shade plant diversity within different agroforestry systems (AFS) at large spatial scales. Using compiled plant inventory data  encompassing four AFS  across six countries in Central America we estimated different metrics of diversity to assess the conservation value of different AFS for shade plants. 458 shade plant species were recorded across the four agroforestry systems. Primary forest species accounted for 28% of the shade species recorded, but only 6% of the recorded individuals. No single AFS was consistently the most diverse across countries when considering rarefied species richness. Trees on pastures can potentially reach a similar species richness as cocoa and coffee systems but require sampled areas 7–30 times larger. In terms of composition, 29 species were shared across the agroforestry systems in different countries, illustrating the strong selection pressure of farmers for species that provide timber, firewood, and fruit. Our study highlights the potential contribution and limitations of different AFS for tree diversity conservation within agricultural landscapes. Most land in Central America was originally forested, however, this forest has undergone a continuous transformation since pre-Columbian times3. Currently, agricultural land in Central America covers ~ 51% of the total land area while natural forests cover only 36% (data from 2010 to 20164).The future of biodiversity conservation in the highly modified landscapes of Central America will depend, in large measure, on how agricultural landscapes are managed. Central America is a region of high conservation value due to its unique mixture of flora and fauna from the Nearctic and the Neotropics and includes some of the most biodiverse ecosystems in the world5, the presence of shade plants on farms can also help to conserve plant and animal diversity7. By ‘shade plant’, we refer to woody vegetation including trees and shrubs; large monocots (i.e. Musaceae and Arecaceae), and in a few cases large tropical herbs and vines  that are used to provide shade to crops and pastures. Diverse agroforestry systems  can help conserve biodiversity by increasing plant density and diversity, providing habitat and resources for wild species, and enhancing the functional connectivity of agricultural landscapes11. For example, trees in agricultural landscapes can create biological corridors among natural reserves and/or remaining forest fragments, facilitating the colonization, movement, and survival of forest species13.While the remaining forests are critical for biodiversity conservation in region8. Multiple studies have shown that, even if the local conservation value of AFS is generally much lower than that of native forests15, AFS can harbor a high richness of terrestrial and epiphytic plants, fungi, birds, mammals, reptiles, amphibians, and insects20. Within AFS, farmers plant, retain from the natural regeneration or simply tolerate21 multipurpose plants that provide food, traditional medicines, timber, firewood, and important ecosystem services25. However, since the natural regeneration of primary forest tree species is often limited within agricultural fields26, over the long term, plant diversity within agricultural landscapes can decline if remnant tree species are not replaced through adequate management of natural regeneration27.The establishment of diverse agroforestry systems (hereafter abbreviated as ‘AFS’) is considered one of the most promising options for maintaining tree cover and conserving diversity and connectivity in agricultural landscapes while maintaining agricultural production31 that either considered how diversity changes across time within a particular AFS32 or lumped different AFS in a single class5. However, AFS are highly variable in terms of their spatial and temporal arrangements, composition , and management practices. As such, comparisons of tree diversity across multiple types of AFS and multiple countries are still needed.Despite growing interest in agroforestry, there is still limited information on the patterns of plant abundance, density, and diversity across different AFS. To date, patterns of plant diversity in AFS have been mainly assessed through local studies focused on a particular type of AFS in one or a few sitesA)Do agroforestry canopies harbor woody species of conservation value? For this, we assess the patterns of abundance of shade plants  in different AFS in Central America.B)How diverse are the canopies of different agroforestry systems? For this, we compare the expected number of species under different agroforestry systems to assess whether there are systems which consistently have higher species richness irrespective of the country where they are sampled.C)How similar are AFS in Central America in terms of species composition? To explore this, we assess to what extent different AFS overlap in shade plant species composition and assess which are the most common shade species found in agroforestry systems.The objective of this article is to characterize patterns of shade plant diversity in four agroforestry systems in Central America, including shaded coffee systems, shaded cocoa systems, dispersed trees in pastures, and live fences, to better understand the ability of AFS to contribute to tree diversity conservation efforts. More specifically we address the following research questions related to the conservation value of agroforestry systems:33 and is home to human communities where agroforestry practice is embedded in cultural practices34. For instance, the production of coffee and cocoa in the region is almost entirely managed under shade35, although levels of shade vary significantly across countries and landscapes36. Trees in pastures and live fences are a conspicuous feature of pastures, providing numerous benefits for ecosystem services and livelihoods30. The maintenance and expansion of agroforestry are encouraged by national governments, coffee institutes , development programs , and research organizations  active in the region. In this context, understanding how agroforestry systems contribute to the conservation of shade plant diversity is of prime importance. The results of this study offer information about both the potential windows of opportunity and limitations of leveraging shade plants on farms for biodiversity conservation.Central America is one of the regions with the largest tree cover outside forests. Across the dataset, 68.8% of individuals were identified to species level, 26.6% were identified to the genus level, 4.4% were identified only with vernacular names, and 0.3% were unidentified. Of the individuals identified at species and genus level , 72.8% were trees, 22.5% were plantains or bananas (Musaceae), 4.4% were palms (11 species in Arecaceae), and 0.3% corresponded to small shrubs (19 species), woody monocots, large herbs, and climbers (Musaceae). The ten most abundant families (in order of importance) were Fabaceae , Musaceae (22.5%), Boraginaceae (11.9%), Malvaceae (6.5%), Bignoniaceae (4.5%), Arecaceae (4.4%), Meliaceae (3.6%), Rutaceae (2.9%), Burseraceae (2.5%) and Anacardiaceae (2.2%), which together accounted for 85.6% of all individuals. There were 18 families represented by ten or fewer individuals in the whole sample , coffee agroforestry systems (COFFEE-AFS), dispersed trees in pastures (DTP), and live fences (LF) across six countries: Panama (PAN), Costa Rica (CRI), Nicaragua (NIC), Honduras (HND), Guatemala (GTM) and Belize (BEL)climbers . Across climbers . Exotic e sample .Musa spp. (22.5%), Cordia alliodora (11.5%), Gliricidia sepium (9.0%), Guazuma ulmifolia (4.5%), Tabebuia rosea (3.3%), Cedrela odorata (2.6%), Bursera simaruba (2.4%), Inga spp. (2.0%), Bactris gasipaes (1.8%), and Citrus maxima (1.5%). At the tail end of the abundance distribution, 50 shade species had only one individual, and 27 species had two individuals in the whole sample (plus another 20 species that were only identified to genus level). In terms of frequency, the average number of plots in which a species occurred was 29.8 plots . The species with the highest frequency of occurrence was Cordia alliodora . Other frequent species included Cedrela odorata (present in 649 plots), Gliricidia sepium (574 plots), Tabebuia rosea (522 plots), and Guazuma ulmifolia (516 plots). 108 shade species were present only in one plot (including 21 which were identified only to genus level).Across all AFS, the ten most abundant species accounted for 61.2% of all individuals. These species were (in order of abundance): Successional guild information was retrieved for 382 woody species ; there were nine species for which we could not retrieve any information. Of these 382 native species, 28% corresponded to intermediate and primary forest species, 37% were species from secondary forests and 10% were species whose forest successional stage was unclear see . The rems100) and 5,000 individuals (Ri5000). There was no clear pattern of one AFS being consistently more diverse than another across different countries, as species richness for each AFS depended strongly on the country of study  and COCOA-AFS , even below LF  followed by COCOA-AFS (Ri5000 = 107), LF (Ri5000 = 92), and finally COFFEE-AFS (Ri5000 = 58).We assessed expected species numbers (here onwards referred to as ‘richness’) using rarefaction curves based on samples (plots) and individuals Fig. . RarefacS100 = 179) had the highest species richness followed by DTP (RS100 = 163) and COCOA-AFS (RS100 = 148). DTP reached these levels of species richness with a total sampled area ~ 7.5 times larger than COCOA-AFS and ~ 12 times larger than COFFEE-AFS. LF was the system with the lowest species richness (RS100 = 115). Considering the number of species per 5,000 individuals, COFFEE-AFS (Ri5000 = 205) had the highest species richness. Species richness in COFFEE-AFS was significantly different from both COCOA-AFS (Ri5000 = 164) and DTP (Ri5000 = 158) which were not different from each other. Again, the least diverse system was LF (Ri5000 = 135).In Nicaragua, considering estimates per sample, COFFEE-AFS (Rs100 = 182) had the highest species richness. Unlike Costa Rica and Nicaragua, these results were obtained in HND with a total sampled area of 43.5 ha for DTP, a value that is between the total sampled area of COCOA-AFS (89.3 ha) and COFFEE-AFS  and COCOA-AFS (Rs100 = 133) had intermediate species richness and were not different from each other. Finally, COFFEE-AFS (Rs100 = 108) had the lowest species richness; this was also the AFS with the lowest total sampled area in this country. In terms of individuals, there were no significant differences among the four systems. COCOA-AFS had the highest species richness (Ri3000 = 136) followed closely by COFFEE-AFS (Ri3000 = 133), DTP (Ri3000 = 122), and finally LF (Ri3000 = 121). In Panama, Guatemala, and Belize, comparisons of species richness across different AFS were not possible due to the lack of sufficient data.In Honduras, considering estimates per 100 samples, DTP  explained 0.69 of the spatial distribution of species composition across the 18 AFS × country combinations; while the second axis explained 0.29 , Ficus spp. (n AFS = 15), Inga spp. (n AFS = 14) and Musa spp. (n AFS = 11). The Musa genera had the highest The analysis using the Partial Importance Value Index (IVIp) of species in each of the 20 AFS × country combinations and the Average-IVIp ant Fig. . For ins27. The tree assemblage in AFS is highly modified in comparison to natural forests but most of the tree species were native. Most of the trees in the AFS studied stem from natural regeneration21 rather than the introduction of exotic species by active planting, a trend more common in other regions38. The presence of these native trees within agricultural lands can improve landscape structure and serve as biological corridors for plants and other taxa such as mammals, frugivorous birds, and insects39, thereby enhancing the conservation value of agricultural landscapes.Overall, our study shows that Central American AFS retain a significant number of trees within agricultural fields and pastures. Across the AFS studied, we recorded 458 shade plant species occurring in shade canopies. This is a low number of species if compared to the high diversity found in tropical forests and moderate if compared to similar studies in other AFS (120–424 species) sampled in smaller areasHowever, our study also suggests that the tree composition within AFS is highly modified compared to that of intact forest. In all the AFS the species composition of the shade canopy was skewed towards secondary forest species and tree species that are useful for farmers. For example, although primary forest species accounted for 28% of the woody species recorded, they represented only 6% of the recorded individuals. In contrast, species from open areas and agricultural areas accounted for only 25% of the species, but 70% of all individuals. As such, the value of the AFS for the conservation of forest plant species is clearly much lower than that of natural forests.41, seed dispersal syndromes42 and the presence of pollinators43, as well as farm management practices . The use of fire, non-selective weed control, and uncontrolled grazing contribute to the reduction of tree cover and can have profound negative impacts on trees present in low densities30.At the same time, the low abundance of late-successional forest species illustrates the challenges for the conservation of diversity in the long term. Many of the late-successional forest species were represented by a median of two individuals in the plots where they were present, and 25 late-successional species (out of 50) were represented by only a single tree in the whole dataset. For these species, the loss of solitary trees could have significant negative impacts at the landscape level. Whether these tree species present in low abundance will persist in the AFS over the long term will depend on species-specific reproductive traits such as mating and breeding systemsOur results suggest that there is no single AFS which consistently was the most species-rich across all countries. For instance, the highest species richness in Costa Rica was found in DTP and COCOA-AFS, in Nicaragua in COFFEE-AFS, and Honduras in DTP systems. Conversely, the least diverse systems were COFFEE-AFS in Costa Rica and Honduras and LF in Nicaragua.36. For instance, coffee production systems in Costa Rica and Honduras had consistently lower species richness than silvopastures or cocoa systems in the same countries. Coffee production systems in Costa Rica are generally considered the most intensified in the region45, with farmers managing a simplified shade canopy of useful trees such as Erythrina spp. and Musaceae. In Honduras, extension services have also traditionally promoted a model of coffee production under a simplified canopy of Inga species planted at low densities (8–10 m apart) and pruning of tree branches, leaving a thin canopy 2–3 m above the coffee plants . In these systems, the native forest species are almost completely removed, and tree diversity is low16. In contrast, in Nicaragua, coffee is produced under commercial polycultures with a shade of mainly Musaceae and Erythrina spp., but also under more traditional systems with more diversified shade46.Many of the differences in shade tree diversity across AFS likely reflect differences in crop and shade management practices. In COFFEE-AFS, shade tree diversity is lower in countries that have intensified coffee production and simplified shade composition4 . Given the large extension of pastures with low tree densities, relatively small changes (increasing or decreasing) in tree cover could have profound impacts at the landscape scale.Second, our results suggest that the species richness of DTP can reach that of COFFEE-AFS and COCOA-AFS but requires a sample area that is ~ 10 to 30 times larger than that of COFFEE-AFS or COCOA-AFS. These results indicate that despite their low species density, the species turnover in DTP is high probably due to a high species diversity per individual and variable species composition across DTP plots. Moreover, the diversity patterns of DTP could have important effects at landscape scales since pastures occupy between ~ 29–69% of agricultural land in different countries of Central America while coffee and cocoa (mainly planted under AFS) occupy between 0.5 and 9.6%C. odorata, C. alliodora), fruit trees , fodder trees , and N-fixing trees  which have rapid growth, prolific regeneration and are easy to propagate by seeds  and cuttings . Farmers actively select and retain (and sometimes plant) these tree species because of their value for farm production and their contribution to family livelihoods, in terms of avoided purchases, safety nets, and food and nutritional security23. Moreover, farmers only use part of the total timber, firewood, and fruit resources available in their farms. For instance, in Nicaragua, farmers report a modest timber harvest ratio at the farm level: < 1 tree ha−1 year−147, and 45–60% of fruits are lost (not used or sold) in coffee agroforestry systems in Guatemala and Peru23. Therefore, it should be possible to increase or maintain the tree cover of species of conservation value without reducing the benefits that farmers already derive from their trees.Finally, our results from the analysis of abundance and IVIp values, and the high similarity shown by clustering around the middle of the Axis 1 and 2 of the NMDS analysis for all the systems, suggest that the composition of all AFS is somewhat uniform across systems and countries. There is a reduced set of species that is common across the four AFS and the Central American region. This set of common trees includes timber species .Our study provides four key insights for tree conservation efforts in the region. First, our study suggests that AFS can play a potentially important role in efforts to conserve tree diversity in the region. Although the AFS studied have a highly modified flora , they still harbor a rich diversity of native tree species, retaining more than 480 tree species. While many of the trees within these systems are early successional species, AFS also retain some forest and late successional species which are important for both plant and animal conservation efforts. In addition, shade trees within the AFS provide critical habitats, connectivity, and resources for the conservation of wildlife within agricultural landscapes49. In addition, these programs could enhance farmer access to information and training on timber production, species selection, tree management, and related aspects. Another important aspect is the need to improve farmer access to tree germplasm for native trees, for instance through capacity building to develop local nurseries or free nurseries supported by NGOs or government programs that make it easy for farmers to access germplasm of native tree species50.Second, our study highlights the important effect of farm management and species selection on shade tree diversity within AFS, and the need for active farmer participation in conservation efforts. Programs that seek to conserve biodiversity across Central America’s agricultural landscapes could further enhance the shade tree diversity within AFS by encouraging farmers to retain a greater diversity of tree species and individuals within their agricultural fields and pastures; to diversify the set of tree species that they actively plant and manage as shade; and to increase the share of species of conservation values. To encourage greater conservation action, conservation programs could potentially support farmers to access payments for ecosystem services, carbon credits, targeted premiums for species of conservation value, certification processes, or other types of rewards or compensation52. Such policy changes could become an opportunity for biodiversity conservation if efforts to increase productivity in these systems go hand in hand with the implementation of practices and incentives to maintain a cover of useful trees and increase tree cover of high conservation value species within cattle production areas53. On the other hand, if intensification efforts discourage the use of silvopastoral systems, this could significantly reduce biodiversity conservation and ecosystem services within agricultural landscapes.Third, our study highlights the need to pay more attention to shade species diversity and management within the region’s vast expanses of pastures. Given the large areas that DTP systems occupy in the agricultural matrix, small changes in tree cover or species turnover could have large impacts at the landscape scale. In recent years, the policy agenda has shifted towards intensifying existing pasture areas, while allowing areas of unproductive pastureland to restore forestsFinally, our study highlights the need for greater standardization of methods for characterizing and monitoring the patterns of shade tree abundance and diversity within AFS. Although shade trees are a conspicuous feature of agricultural landscapes, the methods for measuring tree abundance, species richness, and diversity are not standardized, and this limits our ability to assess the conservation value of trees and track changes over time. There is a need for standardized methodologies for assessing the patterns of tree diversity on farms that are cost-effective, easy to apply, scientifically robust, and comparable. The adoption and use of standardized methodologies would allow us to assess the status and changes in tree resources within agricultural landscapes and would provide valuable information for decision-making for conservation efforts.. The scientific names of all plants were updated to the latest accepted names using World Flora Online . We also checked vernacular names for consistency and spelling errors.To assess the abundance, species richness, and diversity of shade plants in different AFS, we compiled data from twenty-three sources that had plant inventory or census information from AFS in Central America . SourcesCoffea arabica L.) or cocoa (Theobroma cacao L.); or are interspersed on pastures (Poaceae); or are planted in lines between agricultural fields. Shade plants generally occupy a higher stratum in the canopy than the main crop and generally have perennial lifespans. Our definition of shade plants includes woody vegetation (trees and shrubs), large monocots , and in a few cases large tropical herbs and vines . The full list of species was assessed to identify crop and shade species, and all data entries  that corresponded to crops such as coffee, cocoa, maize (Zea mays), and cassava ,  were eliminated from the inventories.As the objective of the study was to characterize the diversity of the shade plants in different AFS, we defined what elements of the vegetation could be considered “shade plants” and updated the databases accordingly. For this study, we defined “shade plants” as those that are regularly intercropped with either coffee  individuals. Of the 2,546 plots, only 144 plots had some NO-ID individuals. Across the whole dataset , 95.6% were individuals identified with scientific names (to genus or species level), 4.2% were identified with vernacular names and 0.2% were NO-ID. The management of these NO-ID individuals for the diversity analysis is discussed further in the description of the methods of each diversity index (see below).−1) of shade species per AFS to identify the presence of extreme values. For the specific case of LF systems which were sampled in lines rather than plots, we assumed a lateral width of 2 m (1 m at each side of the fence) to transform fence length to plot area; thus if a fence was sampled in a line of 50 m, the plot area corresponded to 100 m2. There were differences among the studies in terms of the sampling efforts, number of plots, and sampled areas. For instance, in some cases the studies applied inventories in sample plots  within the farmer plots (the crop management units within farms); in other studies, researchers conducted a full census of all species within farmer plots (with variable areas) or a full census of all trees within the farm. In general, data for plot sizes, abundances, and density were strongly right-skewed. In terms of plot size, larger plots were inventoried in DTP, while smaller plots were more common in COFFEE-AFS and COCOA-AFS , peer-reviewed publications and grey literature , we derived information regarding taxonomic families, species origin , growth form and successional guilds from expert knowledge of the authors (for the most well-known species in the region), online databases (e.g. World Flora Online and GBIF rica see ). We use54. We used the method for rarefaction (interpolation) and extrapolation (prediction) with Hill numbers , to standardize samples based on sample size55 and facilitate the comparison of biodiversity estimates from different studies. To account for differences in overall abundance, sample-based richness estimates were rescaled to the number of individuals sampled using the average number of individuals collected in each sample. Sample-based curves were constructed for each AFS × country combination that had at least 19 plots, as this was the minimum number of plots that allowed us to compare the maximum number of curves (below this there were AFS too few plots to build rarefaction curves). We built rarefaction curves for 18 AFS × country combinations with > 19 plots, while DTP-PAN and LF-BLZ were not included due to their small sample size. All species identified by scientific and vernacular names were included in the analysis. We chose to include data with vernacular names to avoid data loss from some AFS where most individuals were identified with vernacular names . All unidentified species were considered as one class (NO-ID). Since curves were constructed for each AFS × country combination, the chance of inflating richness estimates was low because, within a country, vernacular names are more likely to remain similar. Also, in the case of COCOA-AFS-BLZ, vernacular names corresponded only to one study. All curves were constructed up to a maximum of 250 samples (plots) and 10,000 individuals. Estimates per sample consider community structure while estimates per individual estimate richness assuming a random distribution of all species across the individuals in all the samples collected54. Richness within a country was compared at 100 samples and 5,000 individuals for most AFS × country combinations. However, for AFS in Honduras and Belize, comparisons were made at 100 samples and 3,000 individuals to avoid comparing curves extrapolated beyond 2 times the sampling size of the smallest sample within the country which is the maximum limit recommended for species richness by Chao et al. (2014). All comparisons of a given practice across countries were made at 100 samples and 3000 individuals. Richness estimates and curves were constructed using the iNEXT library56 of R 3.1.0 package57.The large differences in sample sizes, plot size, and the number of individuals are a challenge for the estimation of species richness using data from different studies58.Species composition was assessed using ordination methods, applying non-metric multidimensional scaling (NMDS) with Euclidean distances to explore the similarities among AFS in terms of their botanical composition. For this analysis, all species identified with vernacular names were pooled together as “unknown” species and were considered separately from ‘NO-ID species . The number of individuals per species was standardized per area (where density = log(ind + 1)/log(area + 1). In the NMDS, we only obtained the first two axes and for this, we used 1000 randomizations and a tolerance of 0.0001. All analyses were carried out using QecoWe also assessed botanical composition in terms of which species were shared across different AFS × country combinations and which ones were unique. For this, we used presence/absence data of a given species in each system and summed the number of total occurrences across all possible AFS × country combinations. If a species was present in all AFS × country combinations, the sum would reach 20. In contrast, a value of 1 would imply that a species only occurred in one of the AFS × country combinations.Finally, we calculated the average Partial Importance Value Index (IVIp) to assess the relative importance of species across AFS × country combinations. Partial IVI values were estimated by adding the relative density and relative frequencies of species in each of the 20 AFS × country combinations. If a species was not present in a given AFS × country combination, then the IVIp for that AFS × country combination was 0. All species with no ID were considered as one class (NO-ID) and they were included to estimate the relative abundance of species in each plot. The IVIp did not use information about relative dominance since not all plots provided enough information to estimate the basal area of individual plants. Then, the partial IVIs were averaged Supplementary Information.


Calcium-dependent protein kinases (CDPKs) are an important class of calcium-sensitive response proteins that play an important regulatory role in response to abiotic stresses. To date, little is known about the CDPK genes in white clover. White clover is a high-quality forage grass with high protein content, but it is susceptible to cold stress. Therefore, we performed a genome-wide analysis of the CDPK gene family in white clover and identified 50 members of the CDPK genes. Phylogenetic analysis using CDPKs from the model plant Arabidopsis divided the TrCDPK genes into four groups based on their sequence similarities. Motif analysis showed that TrCDPKs within the same group had similar motif compositions. Gene duplication analysis revealed the evolution and expansion of TrCDPK genes in white clover. Meanwhile, a genetic regulatory network (GRN) containing TrCDPK genes was reconstructed, and gene ontology (GO) annotation analysis of these functional genes showed that they contribute to signal transduction, cellular response to stimuli, and biological regulation, all of which are important processes in response to abiotic stresses. To determine the function of TrCDPK genes, we analyzed the RNA-seq dataset and found that most TrCDPK genes were highly up-regulated under cold stress, particularly in the early stages of cold stress. These results were validated by qRT-PCR experiments, implying that TrCDPK genes are involved in various gene regulatory pathways in response to cold stress. Our study may help to further investigate the function of TrCDPK genes and their role in response to cold stress, which is important for understanding the molecular mechanisms of cold tolerance in white clover and improving its cold tolerance. Ca2+ is a common second messenger in the signaling process of eukaryotic cells3. When plants are exposed to different extracellular stimuli, specific calcium signals are triggered and recognized by different calcium sensors, leading to the activation of downstream cascade systems4. Calcium sensors can be grouped into three main classes: calcium-dependent protein kinase (CDPK/CPK), calmodulin and calmodulin-like proteins (CaM and CaML), and calcium phosphatase-like subunit B proteins (CBL)5. Of these, CDPK is a class of serine/threonine protein kinases unique to plants and some native organisms, and due to their specific structure, CDPKs do not require the involvement of calmodulin and can directly sense and respond to calcium signals6–8.Throughout the life cycle of plants, their growth and development are often influenced by a variety of external environmental factors2+ as a signal, mainly found in plants and protozoa9. A typical CDPK molecule consists of a polypeptide chain with four functional regions or structural domains, which are the variable N-terminal domain (VNTD), the Ser/Thr protein kinase domain (PKD), the junction domain (JD), and the C-terminal calmodulin-like regulatory structure with an EF chiral structure10. CDPK is widely distributed in plants and plays various roles in carbon/nitrogen metabolism, cytoskeleton regulation, stomatal movement regulation, growth regulation11, and plant resistance to biotic and abiotic stresses13. As an ancient gene family, CDPK genes are found in protozoa such as Toxoplasma gondii14 and early lower plants such as green algae15, making them ideal for studying plant genetic evolution.Calcium-dependent protein kinases (CDPKs) are enzymes that directly use ATP-activated protein substrates and Ca15. For instance, overexpression of MDCPK1A in tobacco can remove ROS accumulation and regulate the expression of stress-related genes, thereby significantly increasing cold and salt resistance16. In Brazilian rubber, three CDPK genes  were isolated under low-temperature stress the differential expression trend of CDPK1 indicated that these genes might play an essential role in the low-temperature stress tolerance of rubber18. Wang et al.19 identified 29 CDPK genes in tomato and mined the SiCDPK protein with STK-CAMK type protein kinase structural domain and a unique EF-hand type Ca protein-binding domain. Based on the eight CDPK genes cloned in Cavendish banana, MaCDPK3 was found to be a biomarker associated with drought cold and salt stress20. In grapes, 19 CDPK genes were identified, and the gene expression analysis in wild grapes showed high adaptive capacity and high-expression resistance to known adverse environmental conditions21. In strawberries, nine new CDPK genes were identified, and FaCDPK4 and FaCDPK11 genes were found to be involved in the abiotic stress response through interactions with other proteins, such as proteins involved in the ABA-dependent response to plant stress via Ca2+ signaling, especially RBOHs22. Although CDPK gene families in various crops have been increasingly studied15, systematic analysis of the CDPK gene family in white clover has not been comprehensive.Numerous studies from different plant species have confirmed that CDPK genes play an important role in regulating plant growth and responses to various stimuli, including light, hormones, injury, abiotic and biotic stressesTrifolium repens), also known as white cheatgrass, dutch buckwheat, and creeping clover, is a perennial herb of the genus Trifolium in the Leguminosae family23. It is an allotetraploid forage legume used as a forage grass, now widely grown in temperate and subtropical regions24. It has environmental benefits in terms of biological nitrogen fixation and has medicinal properties, including the ability to clear heat, cool the blood, and calm the heart25. The suitable growth temperature for white clover is 19–24°C, when the low temperature comes in winter, it is easy to suffer from cold and wilts and withers earlier than other grass species such as early grassland and tall fescue; white clover turns green late and does not turn green evenly26. Therefore, improving the cold tolerance of white clover has become an important issue. However, the genetic knowledge of white clover as a non-model plant is very poor28. Until 2019, the genome sequence of white clover was published, and the issue about the genetic background of white clover was resolved29. Related researchers were able to study the function of genes at the genome level, which significantly contributed to the genetic improvement of white clover. For example, Ma et al. identified 133 R2R3-MYBs in the genome of white clover and characterized that some TrMYB genes could positively regulate anthocyanin accumulation and low-temperature response in white clover30.White clover  and serine/threonine protein kinase (Pfam ID: PF00069) was downloaded from the Pfam database, and the HMMER  was performed to recognize and confirm the protein domains that were characterized as candidate CDPK proteins32. The annotation information of all candidate CDPK genes, such as genomic location, protein length, and intron number, was obtained from the white clover genome. Finally, all members of white clover CDPK genes were classified into groups based on similar CDPK genes in Arabidopsis.Genomic resource information for white clover was obtained from previous studies published, all files were provided by Stig Uggerhøj Andersen, Aarhus University33. A phylogenetic tree was generated using the Neighbor-Joining method with 1 000 rapid bootstrap repeats using MEGA11 and protein sequences from Arabidopsis34. The TrCDPK genes were classified into different groups and subgroups based on the phylogenetic tree of AtCDPK and TrCDPK sequences.Candidate white clover CDPK protein sequences were multiply aligned using MUSCLE with default parameters35. MEME searching was performed across TrCDPK protein sequencing using the following parameters: (1) any number of repetitions of a single motif; (2) the maximum number of different motifs up to 10 motifs; (3) the minimum motif width with six amino acids, the maximum motif width of a motif with 50 amino acids. All results were displayed with TBtools36.The MEME program was used to identify the conserved motifs in white clover CDPK protein sequences37. The chromosomal locations of CDPK genes in white clover were drawn with CIRCOS software38, while revealed replication between CDPK genes in white clover. The definition of gene duplication is as follows: (1) the alignment length covered >80% of the longer gene; (2) the identify of alignment region is greater than 80%; (3) only one duplication event gene was counted for tightly linked genes. According to the chromosomal location of CDPK genes, tandem duplication and segment duplication can be identified.The information of the CDPK gene family chromosome physical location was extracted from the white clover genome annotation file, and the gene duplications were identified and characterized based on this information by software MCSanX with default parameter39, including 22,894 Arabidopsis genes and 895,000 interactions (links). All proteins from white clover and Arabidopsis were searched twice against each other in BLAST, with e values cut off at 1e-05, and the highest scoring hit was identified as a homolog of the white clover gene or a homolog of the Arabidopsis gene. These two genes were identified as homologous pairs with two BLAST results. Then, the GRN of Arabidopsis was used to construct the GRN of white clover based on the information of the homologous pairs. Sub-networks containing the white clover TrCDPK gene were searched and analyzed, and the results were visualized using Cytoscape software40. Gene ontology (GO) enrichment analysis was performed on the sub-networks using topGO with the threshold level set to 0.05, showing the representation of the most important terms and designating the highly enriched terms as GRN functions, as described in the software protocol41.The gene regulatory network (GRN) information of Arabidopsis was obtained from the AraNet database (V2)42. These expressional data were transformed using the “log2” function and centered using the “scale” function of the R program. Then, all expression data were clustered and plotted using the “heatmap.2” function of the ggplots package. Meanwhile, the TrCDPK gene expression data were extracted and listed in Supplementary Materials.RNA-seq data have been reported that include eight time points in response to cold stress, which has been described in detail in our previous work and can be evaluated using accession number: PRJNA781064. The Salmon software (version 0.12.0) was used to map the RNA-seq reads to the transcript sequences of white clover genomes, and the expression levels  of each gene were calculated by a subroutine of Salmon43. The growth conditions were a light cycle of 14 h at 24°C and a dark cycle of 10 h at 18°C and were irrigated once a day with a half-strength Hoagland solution. After four weeks of growth, they were randomly assigned into four groups for cold stress treatment. We sampled the following four nodes (0 min (control), 30 min, 1 h, and 3 h, respectively) after treatment, working at 4°C. For each group, three samples were randomly selected from the seedlings and pooled into one biological replicate. All samples were frozen in liquid nitrogen and then stored in a −80°C refrigerator. Total RNA was extracted from white clover seedlings at different time points of cold stress (0 min (control), 30 min, 1 h, and 3 h, respectively) at 4°C using the Total Plant RNA Extraction Kit . cDNA was reverse transcribed using the PrimeScript RT kit . Primers were designed using Primer3 (Table S1)44. qRT-PCR was performed using a Light Cycler® 96 system  and SYBR Premix Ex TaqTM II . The program for qRT-PCR was 95°C for 2 minutes, 40 cycles, 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute. Three replicates were performed for each experiment. The relative expression of the CDPK gene was calculated using 2(-∆∆Ct) analysis45.Seeds of white clover cv. Haifa were purchased from Barenbrug China Ltd. Com. . The seeds were germinated and planted in pots of about 10–15 plants each containing perlite and vermiculite (in a 3:1 ratio), as we described previouslyTo identify the CDPK gene family members in white clover, the CDPK protein sequence of Arabidopsis thaliana was used as the reference sequence and then searched in the genome-wide database of white clover. Subsequently, the candidate genes were subjected to protein structure analysis to determine their Protein kinase and EF-hand structural domains. A total of 50 TrCDPK genes were successfully identified from the white clover genome by multiple sequence alignment, based on the amino acid sequence of the white clover CDPK gene family . As showTo further explore the phylogenetic relationship of the CDPK genes in the white clover, an unrooted phylogenetic tree was reconstructed with 50 TrCDPKs using neighbor-joining methods . This unTo investigate the protein structural and functional diversity of TrCDPKs, motif analyses were performed based on the phylogenetic relationship . GeneralTo determine the evolution and expansion of CDPK genes, we used MCScanX and Circos software to construct the distribution of CDPK genes across chromosomes . As showGene regulation networks (GRN) can be an effective method for studying gene function, and we have predicted GRNs of TrCDPK and their interacting genes according to the public interaction database. As shown in To investigate the function of the TrCDPK genes in response to abiotic stress, we used previous RNA-seq data under cold stress to evaluate their expression profile. Cold stress treatment of white clover was performed at 4°C and RNA-seq analysis was performed at eight time points including 0 H, 30 M, 1 H, 3 H, 6 H, 12 H, 24 H, and 72 H. All expressed TrCDPK genes  were collected and their expression values (FPKM) were grouped using violin plots, which showed that TrCDPK genes increased in response to cold stress . In particular, TrCDPK genes were rapidly activated within 30 min and maintained high expression levels in subsequent stages, the results suggested that TrCDPK genes play a key role in the early stage of cold stress. Through clustering of the expression profiles of these TrCDPK genes and displaying them using heat map functions, the result showed that most TrCDPK genes were highly expressed within 30 min in response to cold stress . This fiTo examine the rapid response to cold stress, we performed qRT-PCR analysis of 10 TrCDPK genes at four time points, consisting of 0 h, 30 min (0.5 h), 1 h, and 3 h. The results of qRT-RCR analysis confirmed their rapid and high expression in response to cold stress . The exp46. Studying the response mechanism of white clover to cold stress is an important way to improve the yield and quality of white clover. Plant CDPK genes and their signaling cascades can regulate plant growth and development and are involved in hormone signaling, abiotic stress signaling, and innate immune responses48.Cold stress affected the normal growth and development of white clover and did not allow normal overwintering and re-growth49. CDPK genes have been identified in many species by whole-genome sequencing and in large numbers. There are 34 CDPK genes in the Arabidopsis genome6, 31 CDPK genes in the rice genome50, and 41 CDPK genes in diploid cotton51. In horticultural crops, CDPK has also been identified in several species, such as 29 CDPK genes in the tomato genome52 and 19 CDPK genes in both cucumber and grape53. During evolution, the CDPK gene family has shown a high degree of structural conservation from mosses to angiosperms49. The gene family is generally divided into four subfamilies with varying degrees of divergent amplification, of which subfamily IV has the smallest number of genes and is also the oldest49. In this study, 50 TrCDPK gene family members were identified from white clover using the bioinformatic methods. White clover contains mainly 16 chromosomes and has a genome size of approximately 841 Mb29. This suggests that there is no linear relationship between the number of CDPK gene family members and the genome size of the species, and it is speculated that some TrCDPK genes lost their function and gradually evolved and died out or evolved into other genes during the evolution of white clover due to the pressure of natural selection.Members of the CDPK gene family have now been identified and extensively studied in numerous plants54. In the present study, we reconstructed 414 gene regulatory networks based on public interaction databases. A large proportion of TrCDPK genes have many interacting genes; for example, TrCDPK03 interacts with 31 genes, TrCDPK21 interacts with 39 genes, TrCDPK39 interacts with 26 genes, TrCDPK50 interacts with 46 genes, and so on. Based on the gene regulatory network and gene function annotation analysis, we found that TrCDPK50 regulated the chr12.jg1202 gene and both were enriched in the signal transduction pathway in biological processes. The chr12.jg1202 gene was a CBL-interacting protein kinase 2-like protein (CIPK). Previous studies have found that the CBL-CIPK complex in Arabidopsis can mediate plant responses to various external stresses, including drought, cold, and wounding56. This could suggest that the TrCDPK genes in white clover may respond with the CBL-CIPK complexes to mediate plant responses to cold stress. According to the results of GO enrichment analysis, most of the genes are focused on processes such as signal transduction, cellular response to stimuli and biological regulation. These results also confirm that the function of TrCDPK genes is highly conserved in white clover. Similar insights have been reported in other plants; for example, CDPKs play an important role in abiotic stress tolerance through hormone signaling and reactive oxygen species (ROS) signaling in response to various environmental stresses such as cold, salt, or drought stress in Arabidopsis, AtCPK10 interacts with the heat shock protein HSP1 and is involved in drought tolerance regulation through abscisic acid (ABA) and Ca2+-mediated stomatal regulation Sheen et al. found that the expression of Arabidopsis AtCPK1 and AtCPK2 was significantly increased under drought and salt-induced conditions58, and Arabidopsis AtCPK10 and AtCPK30 were involved in signal transduction in response to ABA and abiotic stresses. These imply that CDPK genes play an important function in plant resistance to the environment, which is consistent with our findings.Increasing reports are showing that CDPK genes play an important role in signal transduction, and plant response to environmental stimuli, especially in response to biotic or abiotic stresses2+ plays a huge role in plant growth and development as a ubiquitous second messenger in the plant signaling system. When plants are stimulated by the environment and growth and development, stimuli triggered by external factors, such as temperature, light, salinity, and osmotic pressure, produce different calcium ion changes. Specific calcium ion receptors can recognize and sense these changes and then play a role in regulating gene expression through a series of cascade responses59. Through research, CDPK genes sense changes in Ca2+ concentration through EF-hand structures, release self-inhibition, activate kinase structural domains, and then transmit information to regulate physiological changes in plants, which are widely involved in plant growth, development, and morphological construction4. In the present study, the expression of TrCDPK family members changed significantly after cold stress, especially in the early stages of cold stress, as shown in Figure S2. It showed a significant upregulation of TrCDPK gene expression after 0.5 h of cold stress. The expression of 32 TrCDPK genes was up-regulated at 0.5 h of cold stress, with TrCDPK4, TrCDPK8, TrCDPK19, TrCDPK30, and TrCDPK49 being the most significantly up-regulated. The results of qRT-PCR also confirmed the expression profile. These results demonstrate the ability of TrCDPK genes to respond rapidly to cold stress. This finding is consistent with other plants; for example, in Solanum habrochaites, Li et al. demonstrated that the expression of ShCDPK5, ShCDPK6, ShCDPK12, ShCDPK 15, ShCDPK26, ShCDPK29, and ShCDPK33 changed significantly under cold stress; after 0.5 h of cold stress, the expression of ShCDPK5 and ShCDPK6 was significantly up regulated 12 MdCDPK genes were able to respond rapidly in Medicago truncatula under cold stress. These genes play an important role in the signaling pathway of CDPK genes in response to cold stress. Besides, OsCDPK13 and OsCPK17 are considered to be important signaling components in rice in response to cold stress In tomato, CPK27 is a positive regulator of cold acclimation and responds to cold stress by participating in the regulation of ABA, ROS, NO, and MPK signaling pathways63. However, the specific molecular mechanism of TrCDPK in response to cold stress is unclear, and more experiments are needed to explore the function of CDPK genes.It is well known that Ca2+ signaling pathway in white clover to adapt to cold stress.In the present study, 50 members of the CDPK gene family in white clover were identified and characterized. The following analysis was performed: gene identification and classification, phylogenetic and motif composition distribution analysis, chromosomal mapping and gene duplication analysis, genetic regulation network analysis and gene ontology annotation, gene expression analysis, and quantitative real-time reverse transcription PCR (qRT-PCR) analysis. The expression profiles of the TrCDPK genes in response to cold stress were assessed from RNA-seq data, which confirmed their regulatory functions in the cold stress response. Additionally, a white clover GRN was reconstructed using Arabidopsis interaction data. The qRT-PCR results directly revealed that the CDPK genes play a crucial role in response to early cold stress in white clover. The identification and systematic study of the CDPK genes in white clover will help to better explore the function of integrating the CaClick here for additional data file.


The retinol level and retinol delivery to the placenta may vary depending on various factors involving the mother and newborn. The present study evaluates the factors affecting retinol levels in newborns and the transplacental retinol passage in preterm newborns.In this prospective cohort study, the retinol and retinol binding protein (RBP) in the umbilical cord blood of 44 preterm infants with a gestation age of <30 weeks were studied. Serum retinol and RBP levels were determined using an enzyme-linked immunosorbent assay, and the rate of transplacental retinol passage was calculated. The demographic data of mothers and newborns, the use of vitamins by the mother, the application of antenatal corticosteroids, and any diseases diagnosed during pregnancy were recorded. An evaluation was made of the retinol, RBP, and other factors of the mother and newborn affecting transplacental retinol passage.A retinol deficiency was identified in 68.2% of the study population. Retinol and RBP levels in umbilical cord blood  were significantly higher than the corresponding levels in the mother . Umbilical cord blood retinol deficiency was more common in the male participants, while the transplacental retinol passage rate was higher in females. The umbilical cord blood RBP was found to be lower in those administered antenatal corticosteroids than in those who did not receive antenatal corticosteroids, and median maternal RBP levels were lower in patients with anemia and pregnancy-induced hypertension than in those with no disease.Placental adaptation and contributing factors may vary in populations with severe retinol deficiency. The finding of significantly increased cord blood retinol levels when compared to maternal retinol levels in the present study suggests that some compensatory mechanisms, such as increased placental RBP levels, support the presentation of retinol to the fetus, even if the mother has a retinol deficiency. Retinol is an essential vitamin that plays a role in the expression of many genes and in the growth of the fetus. Retinol plays a critical role in pregnancy with regard to rapid cell division and growth ,2. RetinRetinol synthesizes de novo , startinPreterm newborns are known to have lower serum and hepatic retinol levels than term newborns ,15, althThis is the first study in Turkey to evaluate the factors affecting maternal and umbilical cord blood retinol and RBP levels, as well as the rate of transplacental retinol passage in preterm newborns. Determining the retinol levels and the associated factors in the pregnant population in every country, and even in every region, is important for public health.The present study evaluates the factors affecting umbilical cord blood and maternal retinol and RBP levels, as well as transplacental retinol passage in preterm newborns.This cohort study was conducted prospectively between June 2020 and January 2021 in the Obstetrics and Gynecology Clinics of Atatürk University. Ethics Board approval was obtained from the Ethics Board of the Medical School of Atatürk University (Decision no: 2020-7/29). Signed informed consent was obtained from the families prior to patient selection.Included in the study were preterms born in our hospital at gestational week <30, while mothers or newborns with a major abnormality or known congenital metabolic disease, mothers with a chronic liver, kidney or gastrointestinal system disease, and those who did not give consent were excluded.The sex and birth weight of the newborns, and the gestation week, maternal age, gravidity, delivery mode, vitamin intake, vitamin A use during pregnancy, and antenatal steroid treatment in the mothers were recorded. Also recorded were any preterm premature ruptures of membranes (PPROM) diagnosed during pregnancy, as well as preeclampsia/eclampsia, pregnancy-induced hypertension, urinary infection, gestational diabetes mellitus, and anemia, if present.PROM refers to the rupture of membranes prior to the onset of labor, while membrane ruptures before labor and before 37 weeks of gestation is referred to as preterm PROM .Preeclampsia/eclampsia, and pregnancy-induced hypertension were defined according to the diagnostic criteria specified in the current American College of Obstetricians and Gynecologists (ACOG) guidelines .5 colony-forming units/mL of urine during pregnancy were recorded as having a urinary infection [Those with at least one urine culture yielding a bacterial growth of 10nfection .Gestational diabetes mellitus is defined as glucose intolerance diagnosed for the first time during pregnancy based on fasting blood glucose or glucose tolerance tests .Anemia in pregnancy is defined as Hb <11 g/dL in the first trimester, <10.5 g/dL in the second trimester and <11 g/dL in the third trimester .Approximately 6 ml of umbilical cord blood was drawn at birth from all newborns included in the study by experienced individuals using a vacutainer, and the same amount of venous blood was drawn into a biochemistry tube from the mothers’ antecubital region immediately after birth, and centrifuged for 10 min at 4500 rpm after the completion of coagulation, and the sera were separated. The serum samples were frozen and stored at −80 ° until the time of analysis, and were thawed in appropriate ways, with all analyses carried out in the Medical Biochemistry Laboratory of Atatürk University at a single session.The retinol levels of the serum samples were analyzed using ELISA kits obtained from the Bioassay Technology Laboratory , RBP levels were analyzed with ELISA kits provided by Elabscience  following the standard manufacturers protocol and using a Dynex automated ELISA reading device . The measurement ranges of the kits for retinol and RBP were 2–800 ng/mL and 0.07–100 ng/mL, respectively. Both intra-coefficient variability and inter-coefficient variability are < 10%.The formula “umbilical cord blood retinol/maternal retinol ×100” was used to calculate the placental retinol passage rate, identifying the percentage of maternal retinol passing into the umbilical cord blood .Retinol deficiency was evaluated based on WHO guidelines , with a The statistical analysis was carried out using IBM SPSS Statistics v. 23.0 . Descriptive statistics were applied for distributions (number and percentage), and numerical properties for numerical data . Shapiro–Wilk and Kolmogorov–Smirnov tests were used to analyze the normality of the distribution. G-power software  was used to determine the sample size. The number of patients required was determined 42 for t-test analysis of newborns and mother’s groups with 0.55 effect size, 5% type I error, and 80% power. Parametric tests were used to compare maternal retinol and RBP, and umbilical cord blood retinol and RBP levels and the groups with normal distribution of these levels . Nonparametric tests were used to compare retinol and RBP levels in maternal disease groups. A t-test was used for the comparison of parametric tests, the Mann–Whitney U test for the comparison of two groups, and the Kruskall–Wallis test for the comparison of nonparametric data in more than two groups. Pair-wise comparisons were performed using Dunn’s post-hoc test. A Pearson correlation test was used to evaluate whether the continuous variables were correlated. A Chi-square test was used for the comparison of categorical variables with each other. A two-tailed p-value of <0.05 was accepted as significant.During the study period, 76 newborns under 30 weeks’ gestation were born at our hospital, and 44 of these premature newborns who met the inclusion criteria and whose family provided consent were included in the study. Of these, 22 (50%) preterms were female and 22 (50%) were male. The median age of gestation and birth weight were 27.8 ± 2 weeks and 1007 ± 300 g, respectively. The maternal mean age was 26.9 ± 6.5 years and the mothers’ pregnancies were numbered between 1 and 9. Among the mothers, 16 were primiparous (36%) and 28 were multiparous (64%). There was no multiple pregnancy (twins or more) in our study. Three (6.8%) newborns were delivered vaginally. All 41 (93.2%) mothers who had cesarean delivery were administered general anesthesia. Only 3 (6.8%) mothers used vitamins (folic acid and/or multivitamins) for 30–45 days during pregnancy. None of the mothers received additional vitamin A supplements. Among the mothers, 26 were administered antenatal corticosteroids (59%) while 18 were not (41%).The mean maternal retinol and RBP level was 206.4 ± 86.2 ng/mL and 1.04 ± 0.97 ng/mL, respectively, and the mean umbilical cord blood retinol and RBP levels were 273.7 ± 150 ng/mL and 7.88 ± 5.6 ng/mL, respectively. Umbilical cord blood retinol levels and RBP were statistically significantly higher than the respective maternal levels  . A positNo associations were identified between the maternal and umbilical cord blood retinol and RBP levels, nor the birth weight, gestational week or maternal age . No effeIn this study group, the umbilical cord blood and maternal retinol levels in mothers who had received vitamins were similar to those of the mothers who received no vitamin supplements (p:0.23 and p:0.22 respectively). No statistical analysis was made of this group to evaluate any deficiencies, since vitamin use was limited and the duration of vitamin intake was short (30–45 days). Statistical analysis could also not be made of the mode of delivery and the method of anesthesia, since there were only three patients delivered vaginally and all mothers undergoing cesarean section were administered general anesthesia.An analysis of the mothers who had been administered antenatal corticosteroids and those who were not revealed mean umbilical cord blood RBP levels of 5.9 ± 3.07 ng/mL and 10.6 ± 7.3 ng/mL, respectively, being was significantly lower in the group administered corticosteroids (p:0.006) . No diffNo accompanying disorder was identified in 20 of the mothers (45%), while preeclampsia was present in 9 (20%), PPROM in 4 (9%), pregnancy-induced hypertension in 6 (13.6%), urinary infection in 3 (6.8%), and anemia in 2 (4.5%). When the comparison between the umbilical cord blood retinol-RBP and maternal retinol-RBP levels in those diseases was evaluated, maternal RBP levels were found to differ in different maternal diseases, to a statistically significant degree (p:0.02) . In a suThe mean rate of transplacental retinol passage was 134.1 ± 43.5%. A negative correlation was identified between maternal RBP levels and transplacental passage rate . In contrast, no correlation was found between umbilical cord blood RBP levels and the transplacental passage rate , although the mean transplacental passage rate was 148.4 ± 46.8% and 119.7 ± 35.4 % in the female and male newborns, respectively, being significantly higher in the female group (p:0.02). .The detection of retinol deficiency in more than 20% of a society is a serious public health problem, according to WHO criteria . In the In the present study, cord blood RBP levels were also markedly higher than the maternal RBP levels, indicating increased placental RBP in mothers with retinol deficiency. Placental homeostasis has plays a key role in the delivery of retinol to the fetus, and is responsible for the storage of retinoids until the fetal liver maturation is complete ,28. It hSerum retinol levels are lower in preterm newborns than in full-term newborns –32, althDifferent results have been reported on the effect of antenatal corticosteroids on umbilical cord blood retinol levels ,40. IndeRetinol deficiency in the mother may have some negative consequences during pregnancy. In Cochrane’s meta-analysis, it was reported that vitamin A intake, along with iron and folic acid supplementation, may decrease the incidence of anemia in pregnancy in mothers with vitamin A deficiency . RadhikaThis study has some limitations. The high rate of retinol deficiency in our region was an unforeseen condition, and therefore, risk factors such as maternal nutrition were not considered as a cause of retinol deficiency. In addition, due to the COVID-19 pandemic, the number of patients recruited was lower than expected, the study having been planned prior to the pandemic. The numbers were small, particularly in some patient groups, in order to evaluate the effects of maternal diseases on retinol metabolism. Besides, the effect of the mode of delivery and the method of anesthesia could not be evaluated in those who had cesarean delivery Finally, the effect of placental insufficiency on retinol metabolism was not studied, as placental insufficiency was not evaluated.In conclusion, placental retinol passage is regulated according to the presence of a maternal deficiency of retinol. Retinol is essential for the fetus, while excessive amounts are undesirable, and different factors affect its levels in various populations. Significantly high umbilical cord blood retinol and RBP levels when compared to maternal retinol and RBP levels, as was the case in some cases in the present study, suggest that some of the known factors that affect the transplacental retinol passage could be adjusted in populations with maternal retinol deficiencies. That said, studies of the placental compensation in cases of retinol deficiency involving larger populations are required.


Significant increases in brain ceramides and impairments in brain oxygen consumption were observed in the insulin-treated group. These hyperinsulinemia-induced impairments in mitochondrial function were reversed with the administration of myriocin. Altogether, these data demonstrate a causative role for insulin in promoting brain ceramide accrual and subsequent mitochondrial impairments that may be involved in AD expression and progression.Alzheimer’s disease (AD) is the most common form of neurodegenerative disease worldwide. A large body of work implicates insulin resistance in the development and progression of AD. Moreover, impairment in mitochondrial function, a common symptom of insulin resistance, now represents a fundamental aspect of AD pathobiology. Ceramides are a class of bioactive sphingolipids that have been hypothesized to drive insulin resistance. Here, we describe preliminary work that tests the hypothesis that hyperinsulinemia pathologically alters cerebral mitochondrial function in AD mice via accrual of the ceramides. Homozygous male and female ApoE4 mice, an oft-used model of AD research, were given chronic injections of PBS (control), insulin, myriocin (an inhibitor of ceramide biosynthesis), or insulin and myriocin over four weeks. Cerebral ceramide content was assessed using liquid chromatography–mass spectrometry. Mitochondrial oxygen consumption rates were measured with high-resolution respirometry, and H Hyperinsulinemia, both the driving cause and clinical manifestation of insulin resistance , is the Insulin is a hormone secreted by pancreatic β cells that dictates energy utilization within the body and regulates cellular glucose uptake. As a brain growth factor, insulin is necessary in development but is also highly involved in other processes in adulthood . The horAlzheimer’s disease (AD) is the most common form of dementia and is characterized by neurodegeneration that progressively impairs cognition and behavior. The disease affects approximately 6.5 million Americans 65 years and older , which iIn AD, neurodegeneration follows a pattern such that brain regions involved in learning and memory, such as the hippocampus and entorhinal cortex, display neurodegeneration in early disease stages. However, as the disease progresses, neurodegeneration spreads to other cognitive areas of the brain, including areas of the cerebral cortex responsible for language, decision-making, sociality, and other more basic behaviors. Specifically, the connection between the hippocampus and prefrontal cortex is associated with cognitive dysfunction in AD and represents the link between cognition and emotion, which are both disrupted in AD progression . Ceramides are a bioactive family of sphingolipids with structural and functional roles within the cell. Under normal conditions, ceramides found in cell membranes are typically associated with lipid rafts that provide structural support and also have roles in cell signaling that mediate cell growth, proliferation, senescence, and apoptosis, among other processes . HoweverPrevious studies have demonstrated significant elevations in brain ceramide content in patients with AD and other neurodegenerative disorders  and haveThe accumulation of long-chain ceramides has been observed in post-mortem AD brains , and in Over the four weeks of conditioning, insulin injections elicited a significant increase in body weight compared with all other groups at weeks three and four. Myriocin treatment reversed this effect (INS + MYR) a. The chTo identify changes in brain ceramide concentrations following the induction of hyperinsulinemia, a broad spectrum of ceramide species was assessed. Ceramides with side chains C16:1, C20, C24, and C24:1 were significantly increased with insulin treatment and reverted to control levels when combined with MYR . TogetheMitochondrial oxygen consumption rates were measured to assess the effects of hyperinsulinemia on mitochondrial function in our APOE4 mice a. Insuli2O2 production did not vary across the treatment groups  leads to these deleterious and pathogenic changes. Our findings of compromised mitochondrial respiration  in the b2O2 production rate and ceramide content, which are more closely associated with pathology than protection in the context of Alzheimer’s disease [Together, these data add to the growing body of evidence suggesting AD is a metabolic disorder and can be characterized by impairments in brain energy homeostasis and mitochondrial function. More specifically, this work joins other research that suggests ceramides as drivers of AD and potential markers and drug targets of the disease ,39,40. W disease . This wo disease  found th disease , after rCeramides are increasingly recognized as pivotal players in the etiology of myriad cardiometabolic disorders. Indeed, this is so widely acknowledged that some advocate the use of ceramides as a plasma marker of heart disease risk . MoreoveThese findings are especially relevant due to the current widespread nature of metabolic dysfunction and excess adiposity. One study assessing US trends in obesity over recent decades demonstrated that over 50% of young adults (ages 18–25) have overweight or obesity , which hAdult male and female ApoE4 C57BL6 mice (average age of 4.5 months) were group-housed, maintained at 22 ± 1 °C, 60–70% humidity, with a 12 h light–dark cycle, and given ad libitum access to food (LabDiet 5001) and water. Mice were randomly assigned to one of four intraperitoneal injection treatments for 4 weeks: 1. vehicle PBS injections (daily); 2. insulin injections ; 3. myriocin injections in order to inhibit ceramide synthesis ; and, 4. insulin and myriocin injections . At the conclusion of the study, brains were removed, and cerebral cortex tissue was isolated and processed according to subsequent analysis (see below). The following animal studies were conducted in accordance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the IACUC  at Brigham Young University. Additionally, experiments have been reported in compliance with the ARRIVE guidelines 2.0 on reporting animal experiments.After four weeks of conditioning, mice underwent insulin tolerance tests. Mice were fasted for six hours and then received an intraperitoneal injection of insulin (0.75 unit/kg of body weight). Blood glucose was determined at baseline (week 0) and weekly until sacrifice, as indicated in the figures using the Bayer Contour glucose meter .Lipids were analyzed as described previously   with MiR05 respiration buffer as described previously [2 consumption from proton leak (GML). Following stabilization, ADP (2.5 mM) was added to determine oxidative phosphorylation capacity (GMP). Succinate was added (GMSP) for complex III electron flow into the Q-junction. Complex II-supported ETS was then measured by inhibiting complex I with rotenone . Last, residual O2 consumption was measured by adding antimycin A (2.5 M) to block complex III action, effectively inhibiting electron flow. This value provides a rate of respiration that is used as a baseline. Following the protocol, samples were lysed for protein quantification . Respiration rates are shown relative to total sample protein.High-resolution Oeviously . Respiraeviously : electro2O2 generation was measured using an Amplex Red Hydrogen Peroxide/Peroxidase Assay kit  as described previously [2, 1.22 mM MgSO4, and 5.5 mM glucose). The reaction mixture was pre-warmed in a 96-well plate with 100 μL of mixture per well. A 20 μL aliquot of tissue lysate suspended in KRPG buffer (~1.5 × 104 cells) was added to each well. Samples were incubated for 1 h. Fluorescence was measured with a microplate reader .Heviously . A reactp < 0.05. Values are reported as means ± the standard errors of the means. Statistical analyses were conducted by one-way analysis of variance (ANOVA) accounting for the four treatment groups. Tukey’s test was used for comparisons between groups within each experiment, and differences were considered statistically significant at


The periodontal status and distal bone level of the adjacent second molar can be negatively affected by the surgical extraction of an impacted lower third molar. Absorbable materials have some benefits, including enhancing primary wound coverage and promoting wound healing through isolation, clotting, wound stabilization and haemostasis. This study set out to compare primary and secondary healing and collagen-membrane-based primary healing after surgical removal of partially erupted impacted third molars (3Ms), evaluating the distal alveolar bone level (ABL) and periodontal status of the adjacent second molars (2Ms).n = 28), primary healing (n = 27) and membrane-based primary healing (n = 29). Digital panoramic radiographs were obtained preoperatively (T1) and three months postoperatively (T2). The distances between the cemento-enamel junctions and the alveolar bone crests on the distal aspects of the adjacent 2Ms were measured using calibrated radiograph measurement software. The pocket depth and plaque index measurements were performed preoperatively and three months postoperatively. The periodontal plaque index (PPI) scores were registered on the distal aspects of the 2Ms, and the mean values were used.Patients who met the inclusion criteria were randomized into three groups: secondary healing (p < 0.05). In terms of the ABL of the adjacent 2Ms, primary healing (p < 0.05) and membrane-based primary healing (p < 0.05) had superior results to secondary healing.Three of the applied healing types positively affected periodontal pocket depth (PPD) and periodontal index values (Membrane use is promising for the distal bone gain and periodontal status of the adjacent 2M.This clinical study was registered by the Australian New Zealand Clinical Trials Registry, with the trial number ACTRN12618001551280. A serious condition that can arise after the surgical extraction of an impacted lower third molar 3M) is the deterioration of the periodontal status of the adjacent second molar (2M). This condition can take a chronic course that affects the long-term stability and survival of the 2M. A controversial but predominant opinion is that bone loss may occur on the 2M’s distal aspect. In such cases, the 2M’s cementum can be exposed, and the condition may even require tooth extraction –3.M is the Many researchers have focused on factors such as flap design –6, suturHealing type has been suggested to periodontal status of the 2Ms. With regards to partially erupted impacted molars, primary intention healing is only possible with a sliding flap, which would be difficult compared to fully impacted teeth without tissue loss. In these situations, primary wound healing can be supported with various materials .Absorbable materials have some benefits, including enhancing primary wound coverage and promoting wound healing through isolation, clot and wound stabilization and haemostasis . AbsorbaThis study intended to evaluate the following hypothesis: Is the administration of collagen membrane after surgical removal of partially erupted impacted 3Ms effective on periodontal healing? This study compared primary and secondary healing and collagen-membrane-based primary healing after surgical removal of partially erupted impacted 3Ms, evaluating the distal alveolar bone level (ABL) and periodontal status of the adjacent 2Ms. Short-term results of this study were presented in a previous manuscript.This prospective, randomized controlled study included patients with vertical or mesioangular partially impacted 3Ms. Patients were excluded if they had medical illnesses or took medication that could influence the course of postoperative wound healing. The clinical study was registered by the Australian New Zealand Clinical Trials Registry, with the trial number ACTRN12618001551280. Patients between the ages of 18 and 30 were included. Patients were excluded from randomization if they had acute pericoronitis, a pre-existing abscess or cellulites, any restorations or carious lesions on the distal surfaces of the 2Ms, or pathological conditions associated with the 3Ms.Oral contraceptive users and smokers were also excluded.This study’s parameters included gender, duration of surgery, surgical difficulty and the position of the 3Ms. Surgical difficulty was recorded immediately after the procedure and evaluated using a four-class difficulty scale, as follows: 1) extraction requiring forceps only; 2) extraction requiring osteotomy; 3) extraction requiring osteotomy and coronal sectioning; and 4) complex extraction(root sectioning) . The levIn this study methodology, the groups were selected randomly and results were evaluated by another researcher blinded to the groups. Patients were randomized to three groups by the opaque envelope method for blind selection. With this method, a total of 30 Group cards, 10 for each group, was prepared and sealed in opaque envelopes. After each impacted tooth was extracted, one envelope was randomly selected to determine the patient’s operative group. The tissue was closed and sutured according to the patient’s group. These groups were the secondary closure (SC) group, distinguished by partial closure of the extraction site to provide secondary healing; the primary closure (PC) group, distinguished by sliding the flap and suturing primarily to total closure of the extraction site; and the membrane-based primary closure (MBPC) group, distinguished by placing a collagen membrane and sliding the flap to total closure of the extraction site.Surgical operations were carried out by a single surgeon with each patient under local anesthesia, which was achieved with up to4 mL of Articaine-HCl and a 1:100,000 ratio of epinephrine . An incision was made from the anterior border of the mandibular ramus, extending to the distal surface and the buccal gingivodental sulcus of the 2 M. The incision was continuous, with vertical incision oblique into the mandibular vestibular fornix, aligned with the2M’s mesiobuccal cusp. A full-thickness mucoperiosteal triangular flap was elevated. If necessary, osteotomy was performed under constant irrigation.The wound closure was performed with atraumatic silk sutures, depending on the group. In the SC group, the flap was positioned to its former position by a single suture distal to the 2 M, leaving a gap. If necessary, a wedge of mucosa was removed. In the PC group, the sliding flap was sutured. In the MBPC group, the extraction socket was closed with a resorbable collagen membrane , and the sliding flap was sutured primarily.The patients were given standard postoperative medication; antibiotics (amoxicillin + clavulanic acid) for five days, nonsteroidal anti-inflammatory drugs (dexketoprofen trometamol) for 3 days and mouthwash (with 0.12% chlorhexidine) for two weeks, according the body mass of each patient. Additionally, an oral health education intervention was presented. The sutures were removed after seven days.The postoperative measurements and the study’s outcomes were examined and determined by another researcher blinded to the group of patients. One of the outcome variables was the periodontal status of the 2Ms. Periodontal measurements were performed preoperatively (T1) and three months postoperatively (T2). The pocket depths and plaque index scores of the2M’s distobuccal (DB), distolingual (DL), midbuccal (MidB) and midlingual (MidL) surfaces were recorded. All periodontal pocket depth (PPD) measurements were performed in millimetres, using William’s periodontal probe .To assess the periodontal plaque index (PPI) scores, the Silness&Löe plaque index was registered on the distal aspects of the 2Ms,and the mean values were used.To evaluate the difference in bone level, which was the other outcome variable, digital panoramic radiographs in standardized position were obtained preoperatively and three months postoperatively by a single panoramic x-ray device. Cemento-enamel junctions (CEJs) were used as reference points, and the distances between the CEJ and the alveolar bone crest on the distal aspect of the adjacent 2M were measured in millimeters using calibrated radiograph measurement software . All radiological measurements were performed twice and independently by another two clinicians who were not involved in this study, and mean values were recorded preoperatively (T1) and three months postoperatively (T2). Among this study’s primary outcomes were the differences (positive or negative) between the pre-and postoperative radiographic measurements.Sample size was specified by using a power analysis. To detect a significant difference at an effect size of 0.69 and power level of 80% with a 95% confidence level, at least 30 patients were required for each group.Statistical analysis was performed using the SPSS statistical software package, version 20.0 . The normalities of the distributions were tested using the Kolmogorov–Smirnov test. The Wilcoxon signed rank test was used for intragroup comparisons. For intergroup comparisons, the Kruskal–Wallis or chi-square test was used, and for post hoc tests, the Bonferroni correction was used.p > 0.05) except in operation time (p = 0.002) . Eighty-four patients completed the study. The sample included 23 men (27.4%) and 61 women (72.6%), resulting in a male-to-female ratio of 1:2.65. Table p < 0.05). MidB and MidL pocket depths were statistically significantly reduced in the MBPC group , but not in the SC or PC group (p > 0.05). Plaque index and oral health positively differed for all three groups (p = 0.000). There were no statistically significant differences among the groups according to intergroup comparisons of PPD and PPI (p > 0.05).Furthermore, mobility of the adjacent second molar was not observed in any of the patients followed in the study.The intragroup statistics, as well as the plaque index scores and probing depths taken preoperatively and three months postoperatively, are listed in Table p = 0.08).On the other hand, there were statistically significant differences in the PC group(p = 0.01) and MBPC group(p = 0.000) .In binary comparisons, although the change in distal bone level scores was statistically significantly superior in the MBPC and PC groups to that in the SC group, there was no statistically significant difference between the PC and MBPC groups (p = 0.053).Table upp = 0.0 and MBPCVarious factors can influence the surgical difficulty and post-operative complications of an impacted lower third molar, such as position, width, bone and soft tissue coverage, associated pathologies, number of roots, root angulation-dilaceration, gender, age, body mass index, surgeon’s experience, contact with adjacent second molar, etc. –18 This p > 0.05).The results showed that all three groups were comparable in terms of periodontal healing and bone level.The healing result of a single site is dependent on a number of factors at baseline that may vary from site to site or person to person .For thisPostoperative complications as pain, swelling, limitation in mouth opening, bleeding, dehiscence, and dry socket were evaluated in a previous manuscript. The results showed that there was no statistically significant difference associated with collagene membrane usage when compared with the primary closure group . Some otIn studies comparing primary and secondary wound healing and investigating the effect of extracting partially impacted 3Ms on the periodontal health of 2Ms,deep periodontal pockets have been reported , 11 Refep < 0.05).Hashemi et al.  and KorkOn the other hand, the scientific data about dimensional bone changes occurring after 3 M surgery distal to the adjacent 2Mswas limited in the studies investigating primary and secondary wound healing.p < 0.05).These results suggest that primary healing methods may be advantageous in terms of ABL. On the other hand, there was a statistically non-significant positive result on bone level between the primary healing and membrane-based primary healing groups (p = 0.053), and these three-month follow-up results are seen as promising for bone gain on the distal aspects of adjacent 2Ms.In line with our findings, Aimetti et al.  evaluateAccording to this study, all three healing types applied in this study positively affected PPD and PPI values. This result may be related to the easier removal of plaque at the 2Ms’ distal aspects after extraction. On the other hand, in terms of the 2Ms’ ABLs, primary healing and membrane-based primary healing had superior results to secondary healing. In our opinion, especially when the patient’s bone level is insufficient, primary healing is advantageous and preferable. Additionally, the use of resorbable collagen membranes is a promising approach after the surgical extraction of impacted lower 3Ms, as it promotes bone regeneration and improves the periodontal status of the adjacent 2Ms.


The mechanism underlying antimicrobial activity of conjugated bile acids against strains of lactic acid bacilli is not well understood. The purpose of this study was to investigate two typical conjugated bile acids (glycochenodeoxycholic acid and taurochenodeoxycholic acid) for their mechanisms of antimicrobial activity against four strains of different species of lactic acid bacilli at the physiological pH of the small intestine of humans. The bacterial cell membrane integrity, transmembrane potential, and transmembrane pH gradient were examined using the fluorescence probes SYTO 9 plus propidium iodide, 3,3′-dipropylthiadicarbocyanine iodide, and 5(6)-carboxyfluorescein diacetate N-succinimidyl ester, respectively. The intracellular ATP levels were measured by the firefly luciferase-based bioluminescence method. It was found that the antimicrobial activity of conjugated bile acids against the strains of lactic acid bacilli is strain-specific, and glycochenodeoxycholic acid showed significantly greater antimicrobial activity than taurochenodeoxycholic acid against the strains of lactic acid bacilli. The conjugated bile acids inhibited the growth of strains of lactic acid bacilli by disrupting membrane integrity, dissipating transmembrane potential, reducing the transmembrane pH gradient, and depleting intracellular ATP. In conclusion, the antimicrobial activity of conjugated bile acids against lactic acid bacilli is a multifactorial phenomenon. This study will provide valuable information for developing strategies to improve the ability of lactic acid bacilli to tolerate bile in vivo. Probiotics are live microorganisms that confer health benefits on the host when administered in adequate amounts. Lactic acid bacteria are one of the most commonly used probiotic microorganisms . RandomiLactic acid bacilli as probiotic microorganisms are typically characterized by their superior ability to survive in the gastrointestinal tract. Therefore, bile tolerance is often considered an important selection criterion for potential probiotic strains of lactic acid bacilli because bile acids in the small intestine have a detergent-like property and inhibit the growth of lactic acid bacilli . BesidesBile acids are synthesized in the liver and constitute approximately 50% of the organic components of biliary bile . FollowiThe tolerance of lactic acid bacilli to bile acids is strain-specific . VariousThe purpose of this study was to investigate the mechanism responsible for the growth inhibition of lactic acid bacilli by two typical conjugated bile acids, GCDCA and TCDCA. The results from this study will provide helpful ideas for developing strategies to directionally regulate and control the bile tolerance of lactic acid bacilli in the human intestine.Lactiplantibacillus plantarum ATCC 14917, Lacticaseibacillus casei ATCC 334, Lacticaseibacillus rhamnosus ATCC 53103 (GG), and Lactobacillus acidophilus ATCC 700396 (NCFM). Cultures of lactic acid bacilli were routinely stored at −80 °C in MRS broth  containing 30% (vol/vol) glycerol. The strains were maintained by subculturing with 1% (vol/vol) inocula in MRS broth supplemented with 0.5 g/L L-cysteine hydrochloride  and then incubating for 16 h at 37 °C. The cultures were subcultured three times in a similar manner prior to use.The four strains of lactic acid bacilli used in this study were obtained from the American Type Culture Collection . These strains include N0 represents the viable counts in the broth at zero time, and Nt and Nc represent the viable counts in the broth with and without the bile acids after anaerobic incubation, respectively.The conjugated bile acids were evaluated for their antimicrobial activity against strains of lactic acid bacilli using our previous method  with ming for 10 min, and the cell pellets were washed twice with physiological saline and resuspended in a 50 mM HEPES buffer (pH 7.3) containing 20 mM glucose to an optical dispersion at 600 nm (OD600 nm) of 0.3. The suspensions were mixed with the same volume of GCDCA or TCDCA (Sigma-Aldrich) solution with a final bile acid concentration of 5 and 10 mM, respectively. Ultrapure water instead of the bile acid solution was used as the control. Cells were harvested after incubation at 37 °C for 1 h, washed twice with physiological saline, and resuspended in the same volume of physiological saline. The bacterial cells were harvested by centrifugation at 12,000× The bacterial cellular membrane integrity was determined by fluorimetry, as described previously  with min4, and 10 mM glucose to an OD600 nm of 0.05. The suspensions (160 μL) were mixed with 20 μL of 5 μM membrane potential-sensitive fluorescent probe 3,3’-dipropylthiadicarbocyanine iodide (DiSC3(5); Aladdin, Shanghai, China) dissolved in 10% dimethyl sulfoxide (DMSO) in the wells of a black 96-well flat-bottom microtiter plate ) of strains ATCC 14917, ATCC 334, ATCC 53103, and ATCC 700396 by 0.17, 0.07, 0.11, and 0.10, respectively, compared with their control cells, while the 5 mM TCDCA treatment significantly reduced cellular ΔpH of these strains by 0.23, 0.08, 0.16, and 0.13, respectively, compared with their control cells. The 10 mM GCDCA treatment significantly reduced the cellular ΔpH of these strains by 0.32, 0.22, 0.23, and 0.34, respectively, compared with their control cells, while the 10 mM TCDCA treatment significantly reduced the cellular ΔpH of these strains by 0.35, 0.16, 0.26, and 0.29, respectively, compared with their control cells.The 5 mM GCDCA treatment significantly reduced cellular ΔpH , while the 10 mM GCDCA treatment significantly reduced intracellular ATP levels of these strains by 91%, 89%, and 98%, respectively, compared with their controls (p < 0.05). Even at a concentration of 2.5 mM, both GCDCA and TCDCA treatments significantly reduced intracellular ATP levels in all strains except ATCC 14917 (p < 0.05), while the 10 mM GCDCA treatment significantly reduced intracellular ATP levels of these strains by 70%, 52%, and 77%, respectively, compared with their controls (p < 0.05). The TCDCA treatment was generally less effective than the GCDCA treatment in reducing intracellular ATP levels in the strains. The 5 mM TCDCA treatment significantly reduced intracellular ATP levels of strains ATCC 334, ATCC 53103, and ATCC 700396 by 60%, 59%, and 71%, respectively, compared with their controls . Consequently, no single pathway was able to significantly affect the growth of all the strains of lactic acid bacilli, and the antimicrobial properties of GCDCA and TCDCA should be attributed to the combination of multiple pathways.This study found that the exposure of conjugated bile acids caused loss of membrane integrity, dissipation of ΔΨ, dissipation or flipping of ΔpH, and depletion of the intracellular ATP pool in the strains. However, the effects of these pathways varied depending on the strains evaluated. Strains ATCC 14917 and ATCC 334 were most sensitive to conjugated bile acids with respect to their cellular membrane integrity, while strain ATCC 700396 was most sensitive to conjugated bile acids with respect to their intracellular ATP levels. It is reported that bile salt hydrolase is an intracellular enzyme in lactic acid bacteria. In this study, the intracellular pH of the strains exceeded 7.0, which is higher than the reported optimum pH for the activity of bile salt hydrolase in lactic acid bacilli ,36. As aBile response is a multifactorial phenomenon; there are many studies on bile acid resistance mechanisms of lactic acid bacilli strains using physiological and biochemical analysis, multi-omics analysis, and genetic engineering. Due to the existence of different bile acid resistance mechanisms, strains of lactic acid bacilli respond differently to conjugated bile acid exposure . This maCell membranes of lactic acid bacilli are composed primarily of phospholipid bilayers, and their hydrophobic tails are located in the opposite direction of the cytoplasm and extracellular fluid . Bile acYersinia enterocolitica and Bacillus cereus, respectively. Intact cell membranes are the basis for the normal physiological activities of bacteria and the main target of environmental stress . GCDCA aThere are numerous critical or essential functions performed by the bacterial cell membrane, which is composed of roughly equal amounts of proteins and lipids. The main membrane lipid components are phospholipids in the form of a bilayer, which constitutes the major boundary outlining the cell cytoplasm and controls the transport and diffusion of numerous small molecules and secreted proteins between the intracellular and extracellular spaces . In the in close to neutral when the pHex is reduced, many lactic acid bacteria, particularly lactic acid bacilli, have developed a different strategy; the pHin of these bacteria decreases as the pHex decreases during growth in order to maintain a constant ΔpH rather than a constant pHin [i was observed in this study. Despite the fact that GCDCA crosses the membrane more easily than TCDCA, it is more difficult to dissociate within the cell due to its higher pKa value [in of strains of lactic acid bacilli. The transmembrane ion concentration gradient provides the required electrochemical energy for cell growth and many other cell processes (such as ATP synthesis). This energy is PMF, consisting of ΔΨ and ΔpH . Changesant pHin . Using tant pHin . A positant pHin . The decant pHin ,52. In aant pHin . HoweverKa value . This exin are believed to be two important mechanisms by which GCDCA and TCDCA dissipated the ΔΨ of strains of lactic acid bacilli.A wide range of bacterial physiology and behaviors are regulated by transmembrane potential, including pH homeostasis, membrane transport, motility, membrane protein conformation, antibiotic resistance, cell division, electrical communication, and environmental sensing . There hin and dissipation of ΔΨ in the strains of lactic acid bacilli means that the two conjugated bile acids resulted in the collapse of PMF in the bacterial cells because PMF = ΔΨ − ZΔpH  [ATP, an energy-carrying molecule found in the cells of all living organisms, captures chemical energy obtained from the breakdown of nutrients (mainly carbohydrates) and releases it to fuel a broad range of cellular processes involved with cellular survival, growth, replication, and division . Lactic onstant) . TherefoThe antimicrobial activity of conjugated bile acids against the strains of lactic acid bacilli is strain-specific. This antimicrobial activity is a multifactorial phenomenon, and many factors were involved in this process, including loss of cellular membrane integrity, dissipation of ΔΨ, decrease in ΔpH, and depletion of intracellular ATP. The contribution of each factor to total antimicrobial activity varied among the strains. Understanding the pathways by which conjugated bile acids exert their antimicrobial activity against strains of lactic acid bacilli is helpful to develop strategies to enhance bile tolerance of strains of lactic acid bacilli in the small intestine of humans. In order to gain a deeper understanding of the molecular mechanisms involved in the above pathways, it is necessary to investigate the effects of conjugated bile acids on gene and protein expression as well as the interactions between conjugated bile acids and proteins in lactic acid bacilli.