Pontoscolexcorethrurus, Amynthasgracilis) belonging to two families  were found in the Ribeira River valley, occurring concurrently. Abundance was low (< 13 indiv. m-2) compared with other banana plantations worldwide, that frequently surpassed 100 indiv. m-2. More than 70 studies reported earthworms from >200 banana plantations in 28 countries, and mean species richness was 2.7 per site, ranging from 1 to 10 species. Exotics predominated in most sites and P.corethrurus was the most prevalent species encountered. Overall, more than 104 species from 10 families were reported, with around 61 native and 43 exotic widespread species, mainly of the Megascolecidae, Lumbricidae and Acanthodrilidae families. Richness was highest in India (27 spp.) and the Canary Islands (25 spp.), but native species dominated only in a few countries and sites, while exotics were prevalent especially in island countries and Brazil. Lower-input practices appear to be important for earthworm communities and banana plantations can have large earthworm populations in some cases, which may be contributing to soil processes and plant production, topics that deserve further attention. However, many important banana-producing countries have not yet been evaluated, so further work is warranted, both in terms of applied ecology and biodiversity.Bananas and plantains are major commodity/food crops that represent an important habitat for earthworms, although so far, no review is available on earthworm communities associated with banana/plantain crops worldwide. The Vale do Ribeira region is among the largest banana producing areas in Brazil, but little is known of the earthworms living there. Hence, the present study assessed earthworm populations and species in three banana plantations and adjacent Atlantic forest fragments along the Ribeira de Iguape River using standard (hand sorting) methodologies. Furthermore, we review earthworm populations reported in banana/plantain plantations worldwide. Only two species ( Musa, that evolved in Indochina and Southeast Asia, but with major secondary diversification in Africa, India and the Caribbean (LAC) region account for around 25% of the world’s banana production  and root nematode infestation, as well as Sigatoka  Programme (COFM) at Embrapa Forestry in Colombo, Brazil. The earthworm data obtained were used to determine the total species abundances  and richness, per site and land use .Earthworms were collected using an adaptation of the standard sampling method proposed by the Tropical Soil Biology and Fertility (rogramme . In eachrogramme . The matMusa (genus), Musaacuminata, Musabalbisiana, banana, banane, banano, plátano and plantain. These were then crossed with the common names of earthworms in these languages: earthworms, minhoca, oligochaeta, oligoqueta, vers de terre and lombriz de tierra. Online scientific databases Web of Science, Science Direct, Scielo, google academic and the Base de Dados de Teses e Dissertações (BDTD – Thesis and Dissertation Database) of Brazil were consulted. All the resulting publications were consulted and those containing data on earthworm abundance (density and/or biomass) or species identification were selected and these data extracted, as well as information on sampling sites . Earthworm species were separated into different families and into native or exotic to the region of occurrence, and species richness per site and for each group , when available. Although we treated bananas and plantains separately when possible, for most of the analysis we considered them together, since not all publications provided details regarding the types of bananas cultivated, and even plantains are often called ‘bananas.’ Details on the species and management data obtained and presented in this paper are available for download online from the open access repository Mendeley Data at http://dx.doi.org/10.17632/p8ywsnj8c5.1  and biomass (fresh mass in gm-2) were calculated per sampling site (plantation), using data from the present study. When quantitative data from the literature was available for the individual site, it was used as is. When only means for several plantations in the same general location were provided, these were also used. As the interest of the present study was more at the spatial (site-level) rather than the temporal scale, when samples were taken on multiple occasions, and individual means per sampling date were not available, overall means were used. When taken in wet and dry seasons, both values were used as an interval of abundance and biomass (when measured).Quantitative data on the earthworm abundance and biomass obtained from the literature and from the present study were treated as follows. Means of earthworm abundance  corethrurus 24°26'16.85"S, 47°49'31.71"W), 2019, M. Cremonesi, A. Santos colls. COFMBRSP0232, 2 individuals in Atlantic Forest, HMN 389, Registro – SP , 2019, M. Cremonesi, A. Santos colls. COFMBRSP0233, 2 individuals in Atlantic Forest, HMN 389, Registro – SP , 2019, M. Cremonesi, A. Santos colls. COFMBRSP0235, 2 individuals in Atlantic Forest, HMN 389, Registro – SP , 2019, M. Cremonesi, A. Santos colls. COFMBRSP0236, 1 individual in Atlantic Forest, HMN 389, Registro – SP , 2019, M. Cremonesi, A. Santos colls. COFMBRSP0238, 2 individuals in banana field, HMN 379, Registro – SP , 2019, M. Cremonesi, A. Santos colls. COFMBRSP0239, 1 individual in banana field, HMN 379, Registro – SP , 2019, M. Cremonesi, A. Santos colls. COFMBRSP0240, 1 individual in Atlantic Forest, HMN 422, Sete Barras – SP , 2019, M. Cremonesi, A. Santos colls. COFMBRSP0241, 1 individual in Atlantic Forest, HMN 422, Sete Barras – SP , 2019, M. Cremonesi, A. Santos colls. COFMBRSP0242, 2 individuals in Atlantic Forest, HMN 422, Sete Barras – SP , 2019, M. Cremonesi, A. Santos colls. COFMBRSP0244, 1 individual in Atlantic Forest, HMN 422, Sete Barras – SP , 2019, M. Cremonesi, A. Santos colls. COFMBRSP0245, 3 individuals in Atlantic Forest, HMN 422, Sete Barras – SP , 2019, M. Cremonesi, A. Santos colls. COFMBRSP0248, 1 individual in Atlantic Forest, HMN 422, Sete Barras – SP , 2019, M. Cremonesi, A. Santos colls. COFMBRSP0249, 1 individual in banana field, HMN 422, Sete Barras – SP , 2019, M. Cremonesi, A. Santos colls. COFMBRSP0251, 1 individual in banana field, HMN 422, Sete Barras – SP , 2019, M. Cremonesi, A. Santos colls. COFMBRSP0252, 3 individuals in banana field, HMN 422, Sete Barras – SP , 2019, M. Cremonesi, A. Santos colls. COFMBRSP0253, 1 individual in Atlantic Forest, HMN 344, Eldorado – SP , 2019, M. Cremonesi, A. Santos colls. COFMBRSP0255, 1 individual in Atlantic Forest, HMN 344, Eldorado – SP , 2019, M. Cremonesi, A. Santos colls. COFMBRSP0256, 2 individuals in banana field, HMN 344, Eldorado – SP , 2019, M. Cremonesi, A. Santos colls. COFMBRSP0258, 2 individuals in banana field, HMN 344, Eldorado – SP , 2019, M. Cremonesi, A. Santos colls.COFMBRSP0231, 1 individual in Atlantic Forest, HMN 389, Registro – SP , 2019, M. Cremonesi, A. Santos colls.Amynthasgracilis 24°26'54.25"S, 47°49'38.22"W), 2019, M. Cremonesi, A. Santos colls. COFMBRSP0250, 3 individuals in banana field, HMN 422, Sete Barras – SP , 2019, M. Cremonesi, A. Santos colls.COFMBRSP0237, 1 individual in banana field, HMN 379, Registro – SP , 2019, M. Cremonesi, A. Santos colls. COFMBRSP0243, 1 individual in Atlantic Forest, HMN 422, Sete Barras – SP , 2019, M. Cremonesi, A. Santos colls. COFMBRSP0247, 1 individual in Atlantic Forest, HMN 422, Sete Barras – SP , 2019, M. Cremonesi, A. Santos colls. COFMBRSP0254, 1 individual in Atlantic Forest, HMN 344, Eldorado – SP , 2019, M. Cremonesi, A. Santos colls. COFMBRSP0257, 1 individual in banana field, HMN 344, Eldorado – SP , 2019, M. Cremonesi, A. Santos colls.Rhinodrilidae, Megascolecidae) were found at the six sampling sites in the three counties (Table Pontoscolex (Pontoscolex) corethrurus and Amynthasgracilis, both considered peregrine/exotic in southern Brazil , but with some variation between sites  were of n Brazil , especian Brazil . They han Brazil .P.corethrurus in both native forest and banana plantations of the Ribeira River valley indicate that non-native species have extensively colonized disturbed soils of this region. Nonetheless, this potentially widespread occurrence of exotics should be further evaluated both regionally and nationally, in order to better determine the extent of this phenomenon as well as its possible causes.The predominance of -2 and 2.5 to 9 g m-2) tended to be quite low compared with others observed overall in Brazil . This is probably due to the less intensive and more traditional agroforestry management practices used in these sites, including slashing and mulching, as well as the presence of other trees, particularly atmospheric N2-fixing leguminous trees, and the absence of or lower pesticide use , mainly due to the intensive sampling efforts of Talavera in Tenerife  were native and 43 exotic to the sampling sites , mainly due to the higher sampling effort involving a large number of sites in the Canary Islands. However, all of the species encountered on the islands offshore of Africa were exotic, their introduction having been stimulated over centuries of human colonization bringing in exotic soils and crops , 2011. Tethrurus . In Braz, mainly ethrurus  and Mexiethrurus  plantatiSpecies richness in individual banana/plantain fields was measured in 166 of the 210 sites, and was generally very low, with an overall mean of 2.7 species per site worldwide, of which less than one (0.5) was native and 2.1 were exotic , particularly for exotic  species Fig. . AlthougMegascolecidae (22%), Lumbricidae (17%) and Acanthodrilidae (16%) families , Amynthasmorrisi  (5%), Pithemerabicincta  (4%) and Metaphirecalifornica , Perionyxexcavatus Perrier, 1872 and Polypheretimaelongata    and Eiseniaandrei Bouché, 1972 (both with ~4%) and Bimastosrubidus  (3%) being the most frequently reported. Various octochaetid Dichogaster spp. of the Benhamiinae subfamily, i.e., Dichogaster (Diplothecodrilus) affinis , D. (D.) bolaui  and D. (D.) saliens   and the acanthodrilinae Microscolex spp., i.e., Microscolexphosphoreus  with 3% and M.dubius  with 2% of records, were the most reported acanthodrilids. The Dichogaster spp. were found in 11 countries, mainly in Latin America and the Canary Islands, while the Microscolex spp. were found only in the Canary Island banana plantations. Similarly, the ocnerodrilid Ocnerodrilusoccidentalis Eisen, 1878 with 6% of all records, was found in three countries , but most frequently in the Canary Islands.Of the over 100 species found in banana and plantain fields worldwide, most belonged to the families . These wire spp. . The mos2% each) . These mP.corethrurus (11%), found in 15 countries, mainly in Latin America, but also in places as far away as South Africa, India, Bangladesh, Malaysia, Philippines and Taiwan. Interestingly, 37 out of 54 sites (69%) that identified earthworm species reported P.corethrurus as dominant in the banana plantations . Several megascolecids such as A.gracilis are also known to affect soil physical and chemical properties in annual cropping systems  and important (>40%) grain production increases, respectively  in banana/plantain fields, as biomasses over 17 g mectively . Earthwoectively , high stectively , all facectively . This tyectively .-1; Finally, 18 of the major banana-producing countries in the world . However, as many important banana-producing countries have not yet been evaluated, further work is warranted in order to better understand the earthworm communities and their functional roles in plantain/banana fields, and the role of management practices in affecting their populations and diversity worldwide.Earthworms are an important component of banana and plantain fields worldwide and deserve further attention by taxonomists, ecologists and agronomists. Under some conditions, especially in lower-input polycultures, their abundance and biomass may reach high values and contribute significantly to soil processes and plant production. More than 70 studies performed in over 200 banana plantations of 28 countries found >100 species (around 60% of them native) from 10 families, although species richness in each sited tended to be low  and exotic species predominated (particularly


Paracentrotus lividus from two differently contaminated sites along Palermo’s coastline (Sicily) is reported. Analyses were performed on the sea urchins’ coelomic fluids, coelomocytes, gonads or mixed organs, as well as on seawater and Posidonia oceanica leaves samples from the collection sites. PFOA concentration ranged between 1 and 13 ng/L in seawater and between 0 and 794 ng/g in P. oceanica. The analyses carried out on individuals of P. lividus from the least polluted site (A) showed PFOA median values equal to 0 in all the matrices . Conversely, individuals collected from the most polluted site (B) showed median PFOA concentrations of 21 ng/g in coelomic fluid, 153 ng/g in coelomocytes, and 195 ng/g in gonads. Calculated bioconcentration factors of log10BCF > 3.7 confirmed the very bioaccumulative nature of PFOA. Significant correlations were found between the PFOA concentration of the coelomic fluid versus the total PFOA concentration of the entire sea urchin. PERMANOVA (p = 0.001) end Welch's t-test (p < 0.001) analyses showed a difference between specimens collected from the two sites highlighting the potential application of P. lividus as sentinel species for PFOA biomonitoring.The first determination of presence and biodistribution of PFOA in ninety specimens of sea urchin  Perfluoroalkylated substances (PFAS) are a subclass of POPs broadly distributed in the environment due to their extensive usage in a wide variety of applications and products (from food-packaging to electronics)3. Due to the strength and stability of the carbon–fluorine bond, PFAS are generally resistant to hydrolysis, photolysis, or microbial degradation, and are highly persistent in all environmental compartments, especially water4. PFAS have been also found in wildlife6 fish and other seafood9 and have a high affinity for sediments and organic matter due to chemical adsorption phenomena11. Additionally, PFAS can potentially interfere with normal reproductive and hormonal functions as endocrine disruptors, showing adverse effects, for example PFOA exposure is associated with kidney and testicular cancer in humans12.Emerging and persistent organic pollutants (POPs) are a threat to the marine environment and strategies are needed for their periodical and sustainable monitoring13 perfluorooctanoic acid (PFOA) is among the most frequently detected compounds at concentration levels significantly higher than other minor fluorinated pollutants. Probably this situation will continue to be recorded despite PFOA  was added to Annex A of the Stockholm Convention in 2019 and its use has recently been limited by the European legislation14. In this context the monitoring of PFOA in the environment could provide a more reliable evaluation of fluorinated pollutant contamination since PFOA can also result from the degradation of other fluorinated compounds15. Indeed, the widespread occurrence of PFOA-related compounds represents a potential threat for the environment especially to aquatic ecosystems organisms.In the majority of studies reporting PFAS contaminated matrices17. Several marine organisms were investigated for PFOA levels19, although non-invasive sampling is not always possible with all the species and not all the species were indicative of the pollution status of the collection area. In fact, the mobility characteristics of an animal, its sedentarity and longevity are crucial in the approach to biomonitoring studies since an ideal living bioindicator should be strongly connected to the local environment and survive enough time to allow for multiple periodical sampling21.Indeed, PFOA is easily absorbed by aquatic organisms, and its elimination depends on physiological mechanisms that are different among species and sexes23.Moreover, PFOA is present in the sea, with an average concentration from a few units to hundreds of ng/L with higher values in river basins and in the coastal area of industrial cities24, we focused our study on the marine Paracentrotus lividus  widely used in ecotoxicological studies due to its wide distribution, sedentary lifestyle and longevity (about 15 years)25. Additionally, the gonads of P. lividus are consumed as seafood in different Mediterranean countries thus posing, if contaminated, a potential threat to human health26. Moreover, coelomocytes, i.e. the cells freely circulating in the coelom of P. lividus, can be used as biosensors of environmental stress29.In the frame of our research on the presence of POPs in marine animalsP. lividus have been reported30. Conversely, studies on the biodistribution and uptake of fluorinated pollutants by adult P. lividus specimen from the wild marine environment have never been conducted.Recently, the contamination levels of several emerging and persistent pollutants in wild P. lividus, Posidonia oceanica (Linnaeus) Delile, 1813, seawater and brackish water samples collected from the coastal area near Palermo (Sicily).The aim of this research was to investigate the presence and distribution of PFOA in Paracentrotus lividus (45 specimen per each site), 30 l of seawater (15 per site), 40 samples (20 per site) of sea grass Posidonia oceanica  were collected and analyzed together with 30 l of brackish water from site B (15 l from each creek).Three sampling campaigns were carried out at the two sample sites (A and B) on the coast of north-western Sicily Fig. a chosen 31, the choice of sample sites was based on supposedly different status of pollution based on the site position or proximity to human activities .The samplings activity was authorized by the Capitaneria di Porto of Palermo with protocol number: 0029430. In the absence of data about PFOA contamination in the most recent report about chemical contamination in the coastal region subjected to this study32  (90 samples) and coelomic fluid (CF) (90 samples), as well as gonads (G) (63 samples from 32 sea urchins collected in site A and 31 sea urchins collected in site B), or mixed organs (MIX) (27 samples from 13 sea urchins collected in site A and 14 sea urchins collected in site B) consisting of a homogenized mixture of urchin’s inner matrices when gonads were not developed enough for sampling. Due to their mutually exclusive nature the latter two datasets (G and MIX) were merged and labelled as “Gonads or Mixed organs” (GoM) for statistical analysis and graphical representations that needed a uniform dataset of 45 items per site. Further details on the collection of matrices and their labelling are described in the Supplementary Information.25.The size of the sea urchins  ranged between 30 and 51 mm indicating specimen that have lived in their respective site approximately from 3 to 5 yearsMaterials, equipment and software are described in the Supplementary Information.33 to the type of matrix to be analyzed. Recovery percentages (R %) were checked per each batch of analyses by spiking blank samples with different amounts of PFOA analytical standard before the extraction procedure33.PFOA extraction procedures were adaptedspike is the known concentration of spiked PFOA, Dspiked is the instrumental (LC–MS) analytical response of the spiked sample (i.e. the “detected” concentration), Dunspiked is the analytical response of the unspiked sample. R was then used in Eq. Spiked samples underwent the same extraction procedure of unspiked samples and the percentage of recovery R was calculated according to Eq. seawater and [PFOA]creek, which are expressed as nanograms per liter (ppt), all other PFOA concentrations are expressed in nanograms per gram of matrix (ppb).With the exception of [PFOA]2 > 0.99) was recorded in the concentration range from 0.1 to 1000 ppb. The RSDs on three replicates were below 10%. LOD (0.1 ppb) and LOQ (1.0 ppb) were quantified by IUPAC method. LC–MS analyses were performed in the negative ion-monitoring mode (see Supplementary Information).The PFOA standard was used for calibration before each batch of analyses and a linear response  in grams of each matrix  and 2 samples of brackish water (5 l from each creek mouths in site B) were collected for a total of 6 seawater samples and 6 brackish water samples.34.Samples were checked for the presence of PFOA by solid phase extraction (SPE) (see Supplementary Information) followed by LC–MS analysisseawater and [PFOA]creek concentrations (ng/L) were determined from analytical data according to Eq. The percentage of recovery, calculated according to Eq. P. oceanica (20 samples from site A and 20 samples from site B). Each sample was cut in tiny pieces and homogenized using an agate mortar and pestle, weighed (0.5 g) and transferred to a glass tube for extraction (see Supplementary Information).A total of 40 samples of leaves were collected from different individuals of P. oceanica concentrations (ng/g) were determined from analytical data according to Eq. The percentage of recovery, calculated according to Eq. 35. All samples were centrifuged at 4 °C and 1500 rpm for 5 min in a 5804R refrigerated centrifuge  thus separating the supernatant coelomic fluid (CF) from the coelomocytes (CC). CF and CC were then weighed and placed in different glass tubes for subsequent PFOA extractions (see Supplementary Information).The coelomic fluid, containing also the coelomocyte population, was taken from all the ninety collected specimens (45 per site) by inserting an ultrathin and sharp needle (32G 0.26 mm × 12 mm) of a 1 mL syringe through the peristomal membraneCF and [PFOA]CC concentrations (ng/g) were determined from analytical data according to Eq. The percentage of recovery, calculated according to Eq. The extraction of PFOA from 63 samples of gonads (32 from Site A and 31 from Site B) was performed with LC–MS grade methanol following the same procedure used for extraction from CF and CC . In case of undetected PFOA , analyses were repeated for confirmation on concentrated sample extracts.G concentrations (ng/g) were determined from analytical data according to Eq. Twenty spiked samples were prepared from the most abundant samples of gonads (10 spiked samples per site), by adding 25 µL of an aqueous 1 mg/L stock solution of PFOA to 0.25 g of gonads samples. The percentage of PFOA recovery from gonads, calculated according to Eq. In 27 specimens of sea urchins (13 from Site A and 14 from Site B), the developmental status was not sufficient to collect at least 0.1 g of gonad sample. For these individuals, organs remaining after CF and CC collection, mainly intestine and undeveloped gonads, were mixed together and extracted similarly to the other matrices.MIX) samples. The percentage of PFOA recovery from MIX, calculated according to Eq. MIX concentrations (ng/g) were determined from analytical data according to Eq. Spiked samples were prepared by adding 25 µL of an aqueous 1 mg/L stock solution of PFOA to 0.25 g of mixed organs  two levels  and four variables corresponding to the concentrations of PFOA in each type of sample analysed  including the estimated total PFOA concentration. Each term in the analysis was tested by 999 random permutations.A permutational multivariate analysis of variance PERMANOVAFinally, Principal Component Analysis (PCA) (see Supplementary Information: PCA tables and graphs) was performed on a dataset, containing five variables. Specifically sea urchin’s size and PFOA concentrations in each type of sample  as well as in the entire sea urchin (TOT), to verify the multivariate nature of data in a relatively small number of dimensions, thus limiting the loss of information.seawater = 2 ng/L; these values were about one third of those found in Site B  thus supporting the hypothesized difference of the two sites in terms of chemical contamination . Moreover, the median [PFOA]creek = 16 ng/L with a concentration range of 5–34 ng/L suggested that the stream is one of the possible sources of PFOA pollution in Site B.The concentration of PFOA in seawater from Site A ranged from 1 to 4 ng/L with a median value of [PFOA]P. oceanica  . Interestingly, PFOA contamination levels in P. oceanica were three orders of magnitude higher than [PFOA]seawater from the corresponding site, thus suggesting the bioaccumulation capacity of P. oceanica  in each individual collected from Site B was significantly different (p < 0.001) from that recorded in specimens from Site A.Concerning the whole population of Median values of PFOA concentrations were 0  for specimens collected from Site A, while they were 21 (in CF), 153 (in CC), and 73 ng/g (in GoM) for specimens collected from Site B  and the opportunity to use this species as bioindicator of PFOA-contamination.These PFOA concentrations were at least three orders of magnitude higher than those recorded in the seawater from the sampling sites suggesting a high PFOA bioaccumulation capacity of Considering the wide range of recorded concentrations, the average level of PFOA contamination in the various matrices was instead evaluated by calculating the median values of PFOA concentration followed by the range of concentration levels.P. lividus is limited to a few meters per month37, our hypothesis is that high concentrations found sporadically in specimens from site A could be due to episodically contamination (e.g. from grazing of PFOA-containing debris).Such [PFOA] median values in specimens collected from the less contaminated Site A were 0 for all matrices with a wide concentration range of 0–2500 ng/g in CF, 0–250 ng/g in CC, 0–602 ng/g in G, 0–1034 ng/g in TOT due to an extremely limited number of contaminated specimens. Since the home range of 39.On the other hand, in specimens collected from Site B, [PFOA] median values were 21 ng/g in CF (range: 0–3029 ng/g), 153 ng/g in CC (range: 0–629 ng/g), 195 ng/g in G (range: 0–1160 ng/g), and 74 ng/g in TOT (range: 0–2672 ng/g). In this regard, the bioaccumulative potential of PFOA was evaluated by calculating the bio-concentration factor (BCF) according to Eq. 4P. lividus as showed in Table The results (log BCF) obtained were used to assess the bioaccumulation behavior of PFOA in 38, which defines a log10BCF threshold of 3.7, data reported in Table P. lividus matrices.Considering the European Union criterion of bioaccumulability10[PFOA]MATRIX versus the total concentration log10[PFOA]TOT in the specimen . Of all the combinations  with log10[PFOA]TOT  was lower  that generated five principal components (F1–F5) among which the first three represent the system with an 89% of cumulative variability (see Supplementary Information: PCA tables and graphs).The dataset  values Fig. a, and diHowever, while specimens from the less contaminated site are mostly distributed in the negative F1 region .The PCA seems to show a separation between the two sample sites, although without a clear clusterization. Conversely, the PERMANOVA analysis, showed a significant difference between the two sites  confirmed the very bioaccumulative nature of PFOA.This study represents the first assessment of PFOA levels in different matrices of adult sea urchins from wild environment demonstrating both PFOA uptake and bioaccumulation in P. lividus as a bioindicator of PFOA contamination in the marine environment.PFOA content of coelomic fluid (CF) and coelomocytes (CC) correlated with the total amount of PFOA in the sea urchin suggesting non-destructive sampling of these matrices for periodical biomonitoring purposes. Additionally, PERMANOVA and Welch’s test analyses demonstrated that the level of PFOA contamination was significantly different between the two sample sites highlighting the opportunity to employ Supplementary Information 1.


Background and Objectives: In patients with ankle fractures complicated by syndesmotic injuries, no consensus has been reached on the best method of syndesmosis fixation using syndesmotic screws. One previous study revealed no difference in the tibiofibular overlap between two groups with or without syndesmotic screw removal. Other studies have indicated that distal tibiofibular diastasis exists after the removal of syndesmotic screws. In this study, we aimed to confirm the effect of syndesmotic screw removal on diastasis occurrence. We further analyzed the risk factors that may contribute to the widening of the tibiofibular syndesmosis. Materials and Methods: This retrospective study involved a review of the records of 63 patients with ankle fractures accompanied by syndesmosis injuries that required syndesmotic screw fixation. Anteroposterior radiographs were analyzed for each patient at various time points, from syndesmotic screw fixation to outpatient department follow-ups after screw removal. The changes in tibia–fibula overlap (OL), tibia–fibula clear space (CS), and medial clear space (MCS) were analyzed. Further analysis was performed to reveal potential factors that may have contributed to radiographic differences. Results: Compared with the postoperation radiographs following syndesmotic screw fixation, OL decreased (2.0 mm) and CS increased (0.8 mm) in the anteroposterior radiographs from outpatient department follow-ups. No significant changes were noted in OL or CS after syndesmotic screw removal. However, OL decreased (1.8 mm) and CS increased (0.5 mm) before syndesmotic screw removal. No significant change in MCS occurred during the whole observation period. Linear regression analysis did not reveal any significant correlations between potentially related factors and radiographic changes. Conclusions: Marked diastasis had occurred at final follow-up. Notably, the diastasis occurred before rather than after screw removal. This implies that screw removal does not significantly influence the radiographic outcomes of rotational ankle fractures. Patients of ankle fractures with syndesmotic injuries are prone to postoperative diastasis, incongruity of the fibula within the incisure, and posterior translation of the talus ,2. HowevNo consensus has been reached on the best practices of trans-syndesmotic fixation using screws in terms of the selection of the syndesmotic screws, the size of the syndesmotic screws, and the timing of screw removal. Screw removal is still debated due to the uncertainty of its effects on posttraumatic patients with syndesmosis injuries. One study reported that 1 year postsurgery, no statistically significant differences were observed in the tibiofibular clear space between patients with or without syndesmotic screw removal . AnotherIn this study, we aimed to confirm whether diastasis occurred after syndesmotic screw removal and analyze the risk factors related to diastasis of the distal tibiofibular syndesmosis. This study mainly focused on the association between syndesmotic screw removal and radiological changes in the anklesWe retrospectively reviewed all the records of patients with ankle fractures from January 2015 through December 2018 at our hospital. We included patients who (1) sustained Lauge–Hansen classification supination external rotation (SER) or pronation external rotation (PER) ankle fractures, (2) received open reduction and internal fixation of the ankle fracture with syndesmotic screw fixation less than 2 weeks postinjury, and (3) underwent scheduled trans-syndesmotic screw removal. Patients were excluded if they had an open fracture or pilon fracture, if they had a high risk of skin complication, or if they did not undergo syndesmotic screw removal due to advanced age, underlying medical conditions, or personal preferences.Patients underwent operations under anesthesia in the supine position. All the malleolar fractures were reduced and fixed using plates or screws according to the AO Foundation standards. After fixation of the fibula, medial malleolus, and posterior malleolus, syndesmosis stability was assessed based on a lateral stress test or an external rotation stress test using direct visualization or fluorography. Syndesmotic screw fixation was applied in patients with syndesmotic instability or syndesmotic malposition, as determined by preoperational imaging or intra-operative assessment. After reduction, the syndesmotic screws were inserted through four cortices.Ankle range of motion (ROM) exercise and toe touch weight-bearing (TTWB) ambulation were initiated immediately after surgery and continued for 6 weeks. Then, if the radiographic outcomes were acceptable at first-time follow-up around post-operative 6 weeks, partial to full weight-bearing ambulation was instructed in a stepwise manner. Removal of syndesmotic screws was routinely arranged after full weight-bearing on the affected ankle was achieved, around post-operative 10 to 12 weeks. All included patients followed similar rehabilitation protocols without major complications or drop out.Radiographs, which were displayed through Digital Imaging and Communication in Medicine, were retrospectively reviewed for each patient. The radiographs, which were taken pre-operatively, post-operatively, before removal of the screws and at final follow-up, were collected and assessed. All records were examined by a fourteen-year-experienced attending physician (SJC). Intraclass coefficients (ICC) were also analyzed to validate intra-rater reliability. Fracture type was categorized using the Lauge–Hansen classification system.For each standard ankle anteroposterior (AP) radiograph, tibiofibular overlap (OL), tibiofibular clear space (CS), and medial clear space (MCS) were measured. The OL was defined as the distance between the lateral border of the anterior tibial prominence and the medial fibula 1 cm proximal to the tibial plafond . The CS Data analysis of CS, OL, and MCS in anteroposterior radiographs was conducted at three time points: after syndesmotic fixation (post-SF), before syndesmotic screw removal (pre-SR), and at the last follow-up (last-FU). The post-SF radiographs were obtained immediately after syndesmotic fixation. The pre-SR radiographs were obtained immediately before the syndesmotic screws were removed. The last-FU radiographs were the most recent follow-up radiographs that could be obtained after the syndesmotic screws were removed. To compare the difference among the radiographic outcomes at each time point, three periods of interest were defined. The total observation period was defined as the period from the post-SF time point to the last-FU time point. The pre-SR period was defined as the period from the post-SF time point to the pre-SR time point and was equal to the duration of syndesmotic screw retention. The post-SR period was defined as the period from the pre-SR time point to the last-FU time point .t-test was used to analyze the radiographic outcomes of the pre-SR period, post-SR period, and total observation period. Further analysis focused on the significant differences revealed by the paired samples t-test. An analysis of possible related factors , length of pre-SR period, fracture type , and the radiolucent zone around the syndesmotic screws) was performed using a linear regression model to evaluate the associations between radiographic outcomes and each respective factor. Relationships were considered statistically significant at p < 0.05.The data were analyzed using SAS version 9.4 for Windows . A paired-samples Sixty-three patients  with an average age of 46.2 years  who underwent syndesmotic screw fixation were included in this study. The average body mass index of the patients was 25.77 ± 4.73 . Of the indicated injuries, 46 (63.1%) were SER injuries and 17 (36.9%) were PER injuries. Nine of the patients (14.3%) had indicated injuries with dislocation. Syndesmosis fixation was performed with one 3.5 mm syndesmotic screw in all study participants . In the p < 0.001), CS increased by an average of 0.8 mm ± 1.3 mm , and MCS increased by an average of 0.1 mm ± 1.3 mm . The changes in OL and CS in the total observation period were statistically significant. No significant change in MCS in the total observation period was observed. . CS increased by an average of 0.5 ± 1.5 mm , and MCS decreased by an average of 0.1 ± 1.2 mm . The changes in the OL and CS in the pre-SR period were also significant. No significant changes were observed in the MCS (The duration of the pre-SR period was 10.0 ± 0.4 weeks . During  the MCS .p = 0.432), CS increased by an average of 0.3 ± 1.0 mm , and MCS increased by an average of 0.2 mm ± 0.9 . However, the changes in OL, CS, or MCS in this period were not statistically significant  for each parameter analyzed in this study.A pre-SR period analysis was performed to determine the correlation between the radiographic outcomes and the following factors: age, gender, BMI, duration of the pre-SR period, fracture type , and radiolucent zone around the syndesmotic screws. The analysis did not reveal any significant correlations between the radiographic outcomes and the potential influencing factors . AdditioIn this study, we found a reduction in OL and an increase in CS in the total observation period. This indicates that syndesmotic diastasis occurred between the placement of the syndesmotic screw and the final follow-up after screw removal. The period above was defined as the total observation period and was divided into a pre-SR period and a post-SR period. Surprisingly, diastasis was observed in the pre-SR period rather than in the post-SR period. This finding indicates that although diastasis was observed at final follow-up, it occurred before screw removal rather than after screw removal. These results are noteworthy and differ from the results of other studies.Endo et al. used computer tomography to evaluate syndesmotic reduction 2 weeks after syndesmotic screw fixation and 1 year after screw removal . They foIn the study by Jordan et al., all participants underwent screw removal 11 to 16 weeks after syndesmotic fixation, and diastasis was noted after screw removal . This trJordan et al. also found that the MCS did not change significantly in spite of fibula widening . They coWe subsequently investigated potential risk factors that may have been correlated with ankle diastasis during the fixation period. An analysis of correlation was performed between radiographic outcomes and the following potential risk factors: age, gender, BMI, duration of the pre-SR period, fracture type , and the radiolucent zone around the syndesmotic screws. According to the results, none of the potential factors were significantly correlated with the radiographic outcomes of the patients . AlthougThis study had several limitations. First, incomplete radiograph follow-up records may have led to unrepresentative samples, despite the loss of only one data set in the pre-SR period and post-SR period. Nonetheless, the dataset that we collected was sufficient to detect statistically significant differences. Second, we only used standard ankle anteroposterior plain films to investigate the syndesmotic condition. Baek et al. suggested that computed tomography CT should be used to confirm syndesmosis malreduction . HoweverIn this study, we observed that diastasis had occurred at the final follow-up after syndesmotic screw removal. Notably, the diastasis occurred before screw removal rather than after screw removal. This implies that the removal of the syndesmotic screw does not significantly influence the radiographic outcomes of rotation ankle fractures.


The question of whether a first-order liquid-to-liquid transition is at the origin of water’s anomalous properties has been controversial since the pioneering experiments by Mishima et al. in 1985 and molecular simulations by Poole et al. in 1992. Since then, experiments aimed at shedding light on this question have been performed using amorphous ices made from crystalline ice, fueling criticism about their crystal-like nature. In the present study, we avoid crystalline ice at any time of the experiment yet still observe a first-order glass-to-glass transition in vitrified liquid droplets. This makes the strong case for glass polymorphism and the direct thermodynamic connection to the liquid-to-liquid transition at higher temperatures, dismissing the criticism voiced for three decades. 6 K ⋅ s−1 and pressurize the glassy deposit. We observe a first order–like densification upon pressurization and recover a high-density glass. The two glasses resemble low- and high-density amorphous ice in terms of both structure and thermal properties. Vitrified water shows all features that have been reported for amorphous ices made from crystalline ice. The only difference is that the hyperquenched and pressurized deposit shows slightly different crystallization kinetics to ice I upon heating at ambient pressure. This implies a thermodynamically continuous connection of amorphous ices with liquids, not crystals.The nature of amorphous ices has been debated for more than 35 years. In essence, the question is whether they are related to ice polymorphs or to liquids. The fact that amorphous ices are traditionally prepared from crystalline ice via pressure-induced amorphization has made a clear distinction tricky. In this work, we vitrify liquid droplets through cooling at ≥10 Poole et al.  put forw21220Considering the long-lasting debate, it is safe to say that additional experimental evidence is necessary to shed more light on the question. In this work, we study glass polymorphism but avoid starting from crystalline material. Our key question is whether a glass produced directly from the liquid features glass polymorphism or not. If this is the case, the two-liquid model is supported; if not, the view that the first-order transition in amorphous ices represents a transition between distorted ice polymorphs is fostered. To answer our key question, we start from stable liquid droplets and vitrify them using the hyperquenching technique invented in the 1980s by Brüggeller and Mayer . The gla6 K ⋅ s−1 lead to vitrified water of ≥95% purity. The remaining ≤5% may either be vitrified water or stacking-disordered ice I (ice Isd), where the error bar in the calorimetric method and the sensitivity of the diffraction measurements do not allow a distinction .The current state of the art for producing HGW involves liquid micrometer-sized water droplets hitting at ultrasonic speed on a precooled copper plate . Resultitinction , 31. Thatinction A or posstinction B. HGW extinction  just beftinction , 34, whitinction . CalorimStructural Information from X-Ray Diffraction.These glassy samples were then employed to search for glass-to-glass transformations under pressure. d = 0.368 ± 0.003 nm) to 2θ = 30.3 ± 0.2° (d = 0.295 ± 0.002 nm), indicating that HGW has transformed to a much denser amorphous state. That is, the sharp density jump in d = 0.296 ± 0.002 nm, red curve in d = 0.297 ± 0.002 nm). That is, not only HGW and LDA but also d-HGW and HDA are indistinguishable based on X-ray diffraction. This is a very important result, especially considering that the same state is reached from very different starting points. This path independence also implies that the two glasses can be represented using a double well potential, where each glass corresponds to a minimum, which is metastable compared with a crystal. This is exactly the same energy landscape also found in simulations . The heating scans are shown in aled HGW . In othealed HGW . We suggaled HGW  is maskeAfter densification, the thermal behavior of HGW changes substantially. d-HGW prepared at 77 K shows a pronounced exotherm at 119 K that is not present in unpressurized samples (black line in sd) to hexagonal ice (ice Ih) at 180 K, contrasting the case of HDA where it is reported at 225 K  because of incomplete amorphization. The seeds in HGW are more efficient to grow ice Isd—giving rise to an earlier onset and different shape of the crystallization exotherm. Furthermore, this also produces ice I that differs in terms of “cubicity”  . Consequently, also the weak exotherm indicating transformation from ice I (containing cubic stacking faults) to ice Ih  is shifted in the calorimetry scans. While the crystallization exotherm and the polytypic conversion to hexagonal ice are strongly affected through the different ice I impurities in the amorphous matrix, the glass-to-glass exotherm is not at all affected by the presence of these impurities. This makes sense because crystalline impurities do not impede the growth of a glass of low density inside a glassy matrix of high density. In fact, Tonauer et al. have shown that this process involves nucleation and growth of LDA domains within HDA as well as interfaces between LDA and HDA  , 48. Cub and HDA . Also, th, while in simulations, “LDA”  is prepared through ultrarapid cooling of liquid water   , 54. ThePa ⋅ s−1 , 52, 54 Pa ⋅ s−1  in simuleriments . That iseriments . Our experiments  that alleriments B, includeriments . We do neriments B over crh experiences mechanical collapse only for lower temperatures  can be explained as a thermodynamic melting event but not as a mechanical collapse of ice I. This is in agreement with the conclusions by Strässle et al., who show that ice Ieratures . Furthereratures . The quieratures . Very reeratures . At aboueratures . While seratures , 41, 56 eratures . From coSI Appendix, Figs. S3 and S4. After the compression cycles at various temperatures with compression rates of 200 MPa/min . The instrument is equipped with a Goebel mirror, −1. The second heating trace serves as a baseline. The scans were normalized using the melting peak where the value of 6.008 kJ ⋅ mol−1 was used as melting enthalpy of ice.Ex situ DSC was carried out on a DSC 8000 by PerkinElmer. Temperature calibration was done using cyclopentane, adamantane, and indium. About 15 mg of sample was transferred into aluminum crucibles and cold-loaded into the instrument. The samples were warmed from 93 to 298 K, cooled, and again reheated, all with rates of 10 K ⋅ minSupplementary File


The objectives of this study were to investigate temperature distribution at the sheep hoof and evaluate the reliability and diagnostic performance of infrared thermography (IRT) for the detection of footrot and white line disease (WLD) lesions in intensively reared dairy sheep. Hoof lesions were clinically assessed, and IRT was used to measure temperature distribution on hoof superficial tissue in 600 multiparous ewes. Binary regression models were developed and validated, and receiver operating characteristic curves were estimated to assess the predictive value and diagnostic performance of IRT for the detection of hoof lesions. The most sensitive prediction model for the detection of IFR was based on the difference between ambient and hoof heel temperature , whereas the most specific prediction model was based on the difference between ambient and coronary band temperature . In the case of WLD, the diagnostic performance of IRT had limited predictive value. IRT could be a useful tool for hoof health screening in dairy sheep. However, it must be cautiously adapted in cases where environmental, operating, and operator variables are not effectively controlled. Dichelobacter nodosus, whereas the opportunistic presence of Fusobacterium necrophorum deteriorates the severity of lesions [In dairy sheep, foot-related lameness is a significant welfare-challenging issue. Particularly in intensive farming systems, an early, quick, and accurate diagnosis of lameness-related foot lesions has emerged as a field of interest for veterinarians, consultants, and farmers. In these farming systems, footrot and white line disease (WLD) have been recognized among the most significant infectious and noninfectious foot-related lameness causes, respectively ,2,3,4, w lesions . Footrot lesions . WLD ref lesions ,11. Foot lesions ,12,13. C lesions . Subsequ lesions . ConsequThe notion of developing and validating technologies with the potential to serve as point-of-care diagnostics is not new; however, the last two decades has urged the idea of utilizing infrared thermography (IRT) in animal practice ,17. IRT Most of the applications of IRT in animal production refer to the allocation of sites on the body surface with increased temperatures  to detecRecently, IRT has been evaluated as a prediction tool for the diagnosis of foot lesions, mainly in dairy cows ,37,38,39Therefore, the objective of the present study was to describe the temperature distribution at the coronary band, the heel, and the sole of the sheep hoof and evaluate the reliability and diagnostic performance of IRT for the detection of footrot and WLD lesions in intensively reared dairy sheep.Thirty intensive sheep farms with approximately 10,630 ewes in total were included in the study. The farm characteristics were described in detail by Moschovas et al. . In brien = 600) were enrolled in a cross-sectional study from October to December 2020 to record and assess the occurrence, topography, and severity of foot lesions.A multivariable statistical approach  was used to define the typology of the farming systems as the basis for the subsequent selection of representative farms, as detailed by Moschovas et al. . The farThermographic images of the hooves from individual ewes were captured from the dorsal and plantar views almost vertically from a 50-cm approximate distance using an infrared camera . To avoid operator-derived inconsistencies, all the images were captured by the same trained operator using a predefined protocol as regards the settings and the procedure . The thermal sensitivity, noise equivalent temperature difference (NETD), accuracy, emissivity, and resolution of the camera were 0.06 °C, <60 mK, ±2 °C, 0.95, and 320 × 240, respectively. Moreover, to avoid the disrupting effects of direct exposure to the forementioned factors, thermal images were captured inside the barn without exposure to direct sunlight, wind, and increased humidity and always after the gentle removing of debris from hooves. Routine foot-trimming was performed by experienced foot-trimmers and was supervised by a veterinarian who also performed the physical examination, assessed the body condition score , and recorded the foot-health status. Footrot and WLD occurrence were defined by a clinical appraisal of the lesions revealed during trimming. Underrunning of the hoof and necrosis of the underlying tissues, followed by the separation and deformation of the horn, were used to define a footrot case in our study. White line disease was defined as the occurrence of defects at the junction between the abaxial hoof wall and the sole, followed by a various extend separation of the inner layer of the hoof wall from the laminae. All images were processed with Flir Tools software (v 5.X) to record the ambient temperature (AT) and maximum temperatures in three predefined hoof sites: (i) the coronary band (T1), (ii) the hoof heel (T2), and (iii) the hoof sole (T3) . For meaDifferences between the maximum temperatures and AT were calculated by subtraction . Ear tags and lactation numbers of the animals were also available. An Excel database with a total of 4800 records at the hoof level was developed, and an equal number of thermographic images were analyzed.For the statistical analyses, SPSS v23 software  was used, and the statistical significance was set at the 0.05 level. The internal consistency (reliability) of IRT was estimated by calculating the Cronbach’s alpha coefficient, and the dimensionality was estimated using a principal component analysis .ith ewe in the jth farm. Two sets  of three binary logistic regression models (referring to the three hoof sites where the temperature differences were estimated) were used as described below:0 = constant; β1 = coefficient of lactation number (LAC) ; β2 = coefficient of the body condition score (BCS); β3 – β5 = coefficients of DT-1, DT-2, and DT-3 (DTEMP); and ε = random residual error. The backwards stepwise method was used for the selection of the predictor variables in the models, with the p-value being set at the 0.1 level for this purpose.Descriptive statistics were calculated . Data were also analyzed to assess the contribution of the lactation number, BCS, and hoof temperature in predicting the occurrence of footrot and WLD of the 2 indices were used to assess the goodness-of-fit and the amount of variations explained by each individual model. Additionally, the internal validity of the models was evaluated by split-half cross-validation [p-values for the models and predictors were calculated. Receiver operating characteristic (ROC) curves were drawn, and the areas underneath them  were calculated to compare the diagnostic performance of thermal imaging; the optimal efficiency thresholds, as well as the sensitivity (Se) and specificity (Sp) values, when the predicted probabilities were considered for IRT and WLD lesions, were estimated.The Hosmer–Lemeshow (H-L) test, Omnibus test of coefficient, and Nagelkerke Rlidation . The regOverall, the prevalence of footrot and WLD lesions at the hoof level was 1.2% (56/4800) and 7.9% (377/4800), respectively. The mean values of T1, T2, and T3 and of DT-1, DT-2, and DT-3 were 27.9 ± 4.14, 26.2 ± 3.73, and 25.5 ± 3.48 °C and 8.6 ± 3.34, 7.0 ± 2.81, and 6.2 ± 2.60 °C for the coronary band, the hoof heel, and the hoof sole, respectively. In ewes with footrot, the mean values of the maximum hoof temperatures were 30.0 ± 4.48, 28.2 ± 4.15, and 27.2 ± 3.81 °C at the coronary band, the hoof heel, and the hoof sole, respectively a, whereaThe Cronbach’s alpha coefficient was 0.932, indicating a sufficient reliability of IRT for the purposes of the study. Furthermore, only one principal component was found by PCA to have an eigenvalue > 1 (ca. 2.5), accounting for 83.8% of the total variance and, therefore, satisfying the assumption of one-dimensionality of the temperature measurements .2 varied from 0.032 to 0.043 and from 0.012 to 0.013 for the models predicting footrot and WLD lesions, respectively.In all the models, the Hosmer and Lemeshow tests were statistically insignificant, while the Omnibus tests of coefficients were significant, indicating that the models provided a good fit to the data and were predictive, respectively , whereasp < 0.001 for DT-1; p < 0.01 for DT-2 and DT-3), while the lactation number and BCS were not  (p < 0.05); a one-degree increase in BCS was associated with a decreased likelihood of WLD occurrence by 1.75 times.Similarly, in the case of WLD prediction models, the predictive values of DT-1 and DT-3 were significant ( ≤ 0.01) ; a one-dAll the models were successfully validated using split-half cross-validation; one exception was the model that used DT-2 as a predictor of WLD lesion occurrence .Moreover, the c-statistic values varied from 0.668 to 0.689 for the footrot prediction models and from 0.586 to 0.598 for the respective WLD models  and wereThe results showed that IRT is a reliable tool for measuring the temperatures at the coronary band, the hoof heel, and the hoof sole in sheep. Considering the available literature, this is the first study of temperature values at these hoof sites in dairy sheep with thermal imaging that was assessed for its consistency. Another innovative aspect of this study is the use of differences between temperatures at the selected hoof sites and the AT to predict the occurrence of footrot and WLD lesions. The latter confirmed the notion of a high diagnostic value of IRT for detecting hoof lesions. All the models were fairly predictive and were efficiently validated (except for the model predicting WLD lesions using DT-2 as the predictor). However, their performances varied according to the predicted lesions (footrot or WLD) and among the studied hoof sites.Factors that significantly affect the reliability and performance of IRT and, therefore, need to be avoided during thermal imaging of the hoof are: (i) direct exposure to sunlight, (ii) high humidity level, and (iii) convective heat loss , as the radiation measures and thermal imaging thereof are not only a function of the object’s temperature but, also, of its emissivity and conductivity . All theIn the present study, a large database of captured images was developed, and their detailed assessment revealed that the temperature at the coronary band was the highest among the three hoof sites. This was an expected finding, as it is well-known that the coronary band overlies tissues that are rich in vessels to support the increased blood flow demands for the distribution of nutrients in the hoof. Additionally, from the poorer perspiration, the hoof heel and the hoof sole are expected to have lower temperatures due to the outer keratinized horn layer covering themselves; nevertheless, in hooves with footrot lesions, the temperature was increased by almost 2 °C, even in the hoof heel and the hoof sole. On the contrary, much lower temperature differences were observed in hooves with WLD lesions, possibly due to the absence of a noticeable inflammatory response.In general, the suitability of different hoof sites needs to be assessed prior to their consideration in thermal imaging studies in sheep. Currently, relevant studies are scarce, and the mapping of temperature distribution at the hoof is not available. In the real world, uncontrolled environmental conditions, as well as physiological, pathological, and, possibly, genetic factors, influence the hoof temperature and make the suggestion of universally accepted hoof sites for the diagnosis of hoof lesions a complicated task. Another aspect that needs to be considered is the fact that it is not possible to exploit the noninvasive potential of thermal imaging for all the hoof sites. For example, thermal imaging without lifting the feet is possible for the coronary band but not for the hoof heel and the hoof sole.The differences between the studied hoof sites and the AT varied from 6.2 to 8.6 °C. All the studied farms were at plain regions with a temperate climate, while the study was undertaken during autumn. For the use of the developed models of this season, and on a regular basis throughout the year, their across-seasons validation is important in order to determine whether the estimated differences between the studied sites and the AT remain constant; if they are modified, it is likely that the diagnostic performance and applicability of the models are not adequate, and season-specific models need to be developed.Footrot is associated with severe inflammation ,8,10, exThe optimal efficiency thresholds were estimated by estimating the ROC curve coordinates (predicted probabilities) for which the combination of the sensitivity and specificity values were maximized. When the threshold of DT-2 was set at 6.5 °C, the model provided the highest sensitivity for the diagnosis of footrot (ca. 83.0%) but a low specificity (ca. 48.0%); on the other hand, when the threshold of DT-1 was set at 11.3 °C, the specificity for the diagnosis of footrot was ca. 80.0% and the sensitivity ca. 52.0%. Therefore, it can be suggested that a combination of temperature measurements at both the coronary band and the hoof heel is the most appropriate approach for the sensitive and specific diagnoses of footrot. Nevertheless, a limitation in our study, which could have led to an underestimation of the diagnostic performance of thermal imaging for the detection of footrot in the studied hoof sites, is the low prevalence of active footrot lesions; future studies for external validation of the models in farms with a high prevalence of footrot would be of value to address this limitation.For the detection of WLD lesions, the sensitivity of the model using DT-3 as the predictor was ca. 73.0%, whereas none of temperature differences between the studied hoof sites and AT produced satisfying results as regards the specificity of the models. Based on these findings, it could be assumed that the diagnostic performance of thermal imaging at the studied hoof sites is higher for the detection of footrot lesions than for WLD lesions. Severe and extended inflammation of the hoof underlying tissues in the case of footrot is consistent with this finding, whereas, in the case of WLD, the absence of inflammation at the measured sites is likely; this is a finding that adds to the scarce information regarding the pathogenesis of the disease. In any case, the thermal imaging of other hoof regions  could possibly reveal hoof sites with a better diagnostic performance for the detection of WLD lesions. Another finding adding to our knowledge regarding WLD is that the animals with higher BCS had a decreased likelihood of being diagnosed with WLD. This could imply that WLD is associated with the feeding efficiency and the overall nutritional status of animals. However, this is a speculation, and another study design is necessary to confirm this hypothesis and reveal the underlying mechanisms.n = 103 ewes), there was a basic difference in our study design, making further comparisons inappropriate; namely, we assessed the diagnostic performances of hoof temperatures at specific hoof regions rather than the performance of an average hoof temperature (as estimated by drawing a freehand border line to encompass the posterior part of the hooves) [The mean values of the maximum hoof temperatures at the studied hoof sites are consistent with the maximum hoof temperature of healthy ewes as calculated by Byrne et al.  in three hooves) . The sam hooves) . Similar hooves)  assessedFuture studies on the thermal imaging of sheep hooves for the detection of hoof lesions could exploit the knowledge transfer and methodological aspects from studies in other farm animal species, but we cannot extrapolate conclusions from them. The most relevant applications of thermal imaging for the diagnosis of foot lesions have been recently studied in dairy cattle, and its reliability has been sufficiently documented ,38,39,43Increased temperatures in the central and interdigital plantar regions have been observed in dairy cows with digital and interdigital dermatitis  and in cTraining of the thermal imaging camera operator to understand its limitations and the confounding factors is a crucial component for its efficient utilization for diagnostic purposes. This is achievable, as commercial thermal imaging cameras and their software are user-friendly, with low labor and operational demands. Moreover, the benchmarking capability is a key element when considering using thermal imaging to detect hoof lesions; therefore, a single operator needs to handle the camera and interpret the captured images. In our case, the operator was a veterinarian; however, thermal imaging could also be a farmer-friendly technology if the appropriate training is provided. Although the cost of purchasing a thermographic camera is relatively high, the expense can be justified, particularly in large flocks, given that the operational cost is minimal and the benefits from the effective herd health management outweigh the initial expense. In any case, the exploitation of thermal imaging for the detection of sheep hoof lesions does not imply the replacement of clinical examination and exploratory foot-trimming; on the contrary, it offers a rapid, on-farm, noninvasive, low-cost technique for the early detection of candidate animals that need to be further examined for possible underlying hoof pathologies on an evidential basis.IRT is a user-friendly, noninvasive, and remote-sensing technology with the potential to be used on a regular basis for the screening and rapid assessment of hoof health in farm animals. In intensively reared dairy sheep, it provides a reliable and efficient tool for the detection of footrot and white line disease lesions. However, limitations, including environmental, operating, and operator factors, should be considered and appropriately addressed for its most effective exploitation under field conditions. Further studies are needed to elucidate the various thermal imaging application capabilities regarding hoof health assessments. In any case, IRT should be considered a supplementary tool for the early detection of animals with underlying hoof lesions, facilitating their selective physical examination and exploratory foot trimming to reveal and treat hoof diseases.


BamHI catalytic-deficient mutant D94N/E113K was constructed, purified, and shown to bind and protect the GGATCC site from BamHI restriction. The mutant was fused to a 76-amino acid (aa) DNA nicking domain of phage Gamma HNH (gHNH) NEase. The chimeric enzyme was purified, and it was shown to nick downstream of a composite site 5′ GGATCC-N(4-6)-AC↑CGR 3′  or to nick both sides of BamHI site at the composite site 5′ CCG↓GT-N5-GGATCC-N5-AC↑CGG 3′ . Due to the attenuated activity of the small nicking domain, the fusion nickase is active in the presence of Mn2+ or Ni2+, and it has low activity in Mg2+ buffer. This work provided a proof-of-concept experiment in which a chimeric NEase could be engineered utilizing the binding specificity of a Type II restriction endonucleases (REases) in fusion with a nicking domain to generate infrequent nickase, which bridges the gap between natural REases and homing endonucleases. The engineered chimeric NEase provided a framework for further optimization in molecular diagnostic applications.Strand-specific DNA nicking endonucleases (NEases) typically nick 3–7 bp sites. Our goal is to engineer infrequent NEase with a >8 bp recognition sequence. A  The restivities .BsrDI, are part of natural occurring restriction systems. When they are combined with the corresponding small subunits, they become double-stranded (ds) DNA cleaving enzymes -AC↑CGR in NEB buffer 2 (50 mM NaCl) and buffer 3 (100 mM NaCl) supplement with Mn2+ or Ni2+. The fusion NEase also nicks the composite site CCG↓GT-N5-GGATCC-N5 AC↑CGG both upstream (5′) and downstream (3′), potentially generating a long overhang (20 bases) after melting of the duplex. This work demonstrates a proof of concept for fusion of a catalytic-deficient REase and phage gHNH nicking domain and opened the door for further optimization of infrequent NEases in future research and diagnostic applications.In this work, we constructed and purified a catalytic-deficient ft assay . Here, tBsrDI, Proteinase K, pBR322, and pUC19 were provided by New England Biolabs, Inc. (NEB). Synthetic gene blocks were purchased from Integrated DNA Technologies (IDT). Big-Dye Sanger sequencing kit was purchased from Thermo-Fisher/ABI. Restriction buffer compositions (1 ×): Buffer 2: 50 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2, and pH 7.9 at 25°C. Buffer 3: 100 mM NaCl, 50 mM Tris–HCl, 10 mM MgCl2, and pH 7.9 at 25°C. CutSmart buffer (buffer 4 plus BSA): 50 mM Potassium Acetate, 20 mM Tris-acetate, 10 mM Magnesium Acetate, 100 μg/ml BSA, and pH 7.9 at 25°C.Restriction endonucleases, Nb.2+ or Ni2+ divalent cations were supplemented in NEB buffer 2 or 3 as indicated. DNA blunting kit and blunt/TA quick ligation kit were provided by NEB. Escherichia coli competent cells T7 Express (C2566) and NEB 10β were also from NEB.In some nicking reactions, MnBamHI site and nicking site(s) in pUC19 and the adjacent sequences. The primers were purchased from IDT and have the following sequences:Nicking reactions were usually carried out at 37°C for 30–60 min or as indicated in specific buffers. One forward and one reverse primer were used to sequence (map) the pUC-F61, 5′ CAGCTTGTCTGTAAGCGGATGCCG 3′.pUC-R600, 5′ GGGCAGTGAGCGCAACGCAAT 3′.BamHI site is artificially defined as top and bottom strand as following:The DNA strands of the pUC19 sequence near GGATCCCCGGGTACCGAGC…-3′.top strand: 5′-…AGACCTAGGGGCCCATGGCTCG… -5′.bottom strand: 3′-…TCTNdeI and XhoI digested) using Gibson assembly kit (NEB) and the DNA inserts were selected by plasmid DNA transformation and the correct insert was verified by DNA sequencing. Plasmids were prepared by plasmid mini-preparation kits . DNASTAR Lasergene software was used for sequence editing and visualization of nicking sites (doublet peaks) in DNA run-off sequencing.Synthetic gene blocks were assembled into pTYB1 vector  213 aa >MEVEKEFITD EAKELLSKDK LIQQAYNEVK TSICSPI WPA TSKTFTINNTNVYKEFIEKNCNGVVPI KELCYTLLED TYNWYREKPL DILKLEK KKG GPIKTGNISSA HRSMNKLLLG LKHGE IDLAI ILMPIKQLAYENSELKRVGM EFYLTDRVTNFE ELEPYFELTE GQPFIFIGFN AEAYNSNVPL IPKGSDGMSKRSIKKWKDKV ENKBacillus phage gHNH small nicking domain (76 aa)>-fyrskawer lreqalmrdk glclhcknnr kikvadmvdh iipikvdpslklklenlqsl cnpchnrkta edkkkygBacillus phage Gamma HNH endonuclease 127 aa (GenBank accession number YP_338236)>WT .mkkplrpcce fhcynltreryceehrykek etqqdknryy drfkrdkestfyrskawer lreqalmrdk glclhcknnr kikvadmvdhaiipikvdpsl klklenlqsl cnpchnrkta edkkkyg.BamHI mutant gene or BamHI*-gHNH fusion gene in a plasmid was cultured at 37°C to late log phase and enzyme production was initiated by addition of 0.5 mM IPTG final concentration and cells were induced at 18°C overnight in a temperature-controlled shaker. Cells were lysed by sonication in a chitin column buffer  at 4°C. To remove nucleic acids from enzymes, BamHI*-gHNH fusion protein eluted from the chitin column was diluted in a buffer  and passed through HiTrap DEAE column . Enzymes were concentrated by low-speed centrifugation in protein concentrators and resuspended in enzyme storage buffer.The IMPACT protein expression system, including pTYB1 vector and chitin beads, was provided by NEB. Target protein was cleaved from the intein-CBD fusion by DTT cleavage overnight and eluted from a chitin column . Enzymes were further purified by chromatography through a heparin column . Proteins were concentrated by low-speed centrifugation in protein concentrators (10 kDa cut-off) and resuspended in enzyme storage buffer  and kept in a –20°C freezer. T7 Express cells carrying BamHI endonuclease dimer in complex with cognate DNA had been solved previously  with diminished catalytic activity . Purified E111A binding also protects the BamHI site against BamHI digestion although a small fraction of DNA was linearized, and some nicked circular DNA was also generated (lane 5). BamHI alone digested pBR322 to linear DNA (lane 6). The mutant enzyme also blocked SmaI digestion of pUC19 because the BamHI site overlaps with a SmaI site in GGATCCCGGG sequence (data not shown). No BamHI cleavage activity was detected using 4 μg of the double mutant protein to digest pBR322 (data not shown)   . We conc2+ and it has poor activity in Mg2+ buffers (BamHI*-gHNH fusion (BamHI* denotes D94N/E113K double mutant) was cloned into pTYB1 and the fusion enzyme was partially purified by chitin column chromatography and analyzed by SDS-PAGE (BamHI double mutant (D94N/E113K) alone. The purified enzyme is active in nicking of pUC19 in a time course (5–60 min digestion) in NEB buffer 2 supplemented with 1 mM MnCl2 . The full-length phage gHNH endonuclease is fully active in Mg2+ buffer and nicks the sites AC↑CGR (or AC↑CG in over-digestion)  and medium to high salt are required for the chimeric enzyme to minimize non-specific (star) nicking activity (see below).Our goal is to construct a chimeric NEase with infrequent nicking sites. The minimal phage gHNH nicking domain consists of 76-aa residues and has attenuated activity (the WT enzyme has 127-aa residues). The truncated nicking domain is more active in the presence of Mn buffers . A schemSDS-PAGE . The fusmM MnCl2 . It is eMnCl2 (2 . It is aigestion . In an eigestion . Therefogestion) . The speBamHI binding sequence. It is based on the appearance of doublets in sequencing base calling. When the template strand is broken (nicked or cleaved), the sequencing reads with doublet A/C, A/G, A/T, or extra high A peak are generated by the template-independent terminal nucleotide transferase activity of the Taq DNA polymerase [or extra T peak in doublet  in reverse strand sequencing]  is detected near the BamHI site (GGATCC N5 AG↑TCG) . Nicking at this star site was diminished in NEB buffer 3 supplemented with 1 mM MnCl2 or when this site is mutated to CTATA sequence (data not shown). DNA sequences ACCGA or ACCGC further downstream of the BamHI site were not nicked. Some intervening sequences were manually deleted (as marked by Del1 and Del2) to show the downstream nicking sites. This mapping experiment showed that the fusion nicking enzyme recognizes GGATCC as an anchor site and nicks DNA 5–6 bp downstream (3′) or upstream (5′) in the cognate site ACCGR or a nearby site AGTCG. The stand-alone nicking sites  farther away from the BamHI site are poor substrates for the fusion enzyme.We used the same run-off sequencing to determine the nicking site of the chimeric NEase as described above. The schematic diagram of the sequencing reaction is shown on the top of  AG↑TCG) . The samBamHI site, we deleted the BamHI site in pUC19 by treatment of BamHI-linearized plasmid with a blunting kit and religated the DNA and transferred the DNA into NEB 10β by transformation. Individual plasmids were prepared and confirmed the absence of BamHI site by BamHI digestion (data not shown). The pUC19-ΔBamHI was used as a substrate for BamHI*-gHNH nicking digestion. Only a small fraction of the DNA was nicked in 30 min digestion, and a majority of the DNA remained intact as supercoiled form  (data not shown), indicating possible non-specific nicking activity directed by the nicking domain or by some contaminating E. coli nucleases. In a control digestion, the fusion nickase completely nicked pUC19 in Mn2+ buffer although a small amount of linear DNA was also generated. We concluded from this experiment that efficient nicking of a plasmid required the presence of BamHI site and gHNH nicking site. Some non-specific nicking may occur by the gHNH nicking domain during prolonged digestion.To analyze the nicking activity dependence on a BamHI site and a gHNH nicking site. No nicking was detected upstream at TATAG (complement CTATA) after replacing the star nicking site. However, we have not analyzed all possible star sites for the gHNH nicking domain in Mn2+ buffer.To further map composite nicking sites, we constructed two pUC19 derivatives with GGATCC N(4–5) ACCGG and replace the star site AGTCG (complement CGACT) with CTATA (complement TATAG) upstream. The plasmids with one composite nicking site GGATCC N4 ACCGG or GGATCC N5 ACCGG were digested by the fusion enzyme and the nicked DNA was subjected to run-off sequencing. 2+. 2+/Mn2+ and Mg2+/Ni2+ buffers . The bottom strand also contains a nick near the BamHI site  supplemented with 1 mM MnClBamHI mutant D94N/E113K and a phage gHNH nicking domain (∼76 aa). The fusion enzyme nicks near the BamHI site N4-6 when a nicking site ACCGR/YCGGT is present either downstream or upstream or on both sides. The activity on the gHNH nicking site ACCGR or its variants alone is rather low, and no doublets were detected in run-off sequencing of ACCGR sites further downstream or upstream under limited digestion, which is likely due to the attenuated activity of the small nicking domain that requires a strong DNA binding partner to anchor onto DNA. The nicking site recognition could be relaxed to nick star sites (ASYSR) in Mn2+ buffer, which is consistent with the star activity of the full-length gHNH endonuclease in Mn2+ buffer (1–2 base off from ACCGR sites) (2+/Mn2+ buffer or Ni2+ buffer. The fusion enzyme is active in nicking reactions with 50–150 mM NaCl (200 mM NaCl slightly inhibits activity). The fusion NEases is active in Mn2+ or Ni2+ buffer, and it has low nicking activity in the presence of 10 mM Mg2+. To study the nicking site profile of gHNH minimal nicking domain in Mn2+ and Ni2+ buffers, it would be necessary to construct NGS libraries from nicked DNA and sequencing the inserts and map the sites back to the undigested DNA by examination of thousands of sites. The divalent cation Mg2+ is probably not required for specific binding to the BamHI site for the D94N/E113K mutant as the single mutant D94N or E113K can bind to DNA in the absence of divalent cations (EcoRV-specific binding to the cognate site (GATATC), a divalent cation is required  . To mini cations . For mosrequired . For exaEcoO109I, EcoRI, EcoRV, BsoBI, BspQI/SapI, HincII, NotI, PacI, PvuII, and PpuMI)    (AlwI)  , in bactcleases) , in mobicleases) , in Chlocleases) , 2006, acleases) , I-TslI cleases) , I-PfoP3cleases) , reviewecleases) ]. NEasest.BsrDI)  or by ni) (AlwI) .BamHI site. In FokI footprint analysis using methylation protection experiment with dimethyl sulfate (DMS) modification and hydroxyl radical Fe(II)-EDTA foot-printing study, FokI protected ∼3 bp 5′ upstream of its recognition sequence GGATG and some additional bases at the 3′ end (the cleavage domain reaches out and cleaves DNA downstream) (SmaI digestion of pUC19 plasmid with the overlapping sites in GGATCCCGGG (SmaI site = CCCGGG), suggesting BamHI occupies more than 6 bp.It is not surprising that the fusion NEase nicks a few bases (N4–6) downstream of the nstream) . It is vThe infrequent NEase reported in this work might be useful for end labeling and DNA optical mapping when its non-specific nicking activity could be minimized. To enhance specific nicking of the composite site, it may be necessary to incubate DNA substrate with the fusion enzyme for 10–20 min to form enzyme-DNA complex and then add divalent cations to activate nicking for a short period of time (5–10 min). Another possibility is to utilize mutants of the 76-aa nicking domain with further weakened DNA binding affinity. Future study and optimization of the infrequent NEase is required prior to any biotechnology applications.BamHI double mutant may be used in in vivo study of DNA binding proteins overlapping a BamHI site or it can serve as a repressor  and gHNH nicking domain fusion which binds to 5-hydroxylmethylcytocine (5hmC)-modified DNA and introduces nicks near 5hmC modified sites for labeling and detection on mammalian DNA (The cleavage-deficient epressor . As reponear DNA . Similarlian DNA .The original contributions presented in the study are included in the article/S-YX designed and performed the experiments, interpreted the data, and wrote the manuscript.S-YX is an employee of New England Biolabs, Inc. a company developing restriction enzyme and nicking enzyme reagents for the research and diagnostic community.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Function, Gaspers et al.2+ oscillations and Ca2+ waves in the liver. The inhibitory effect of ethanol on Ca2+ signaling is proposed to be due to acetaldehyde formation catalyzed by alcohol dehydrogenase (ADH), because it was blocked when ADH was inhibited and enhanced when the enzyme acetaldehyde dehydrogenase was inhibited.2+ signal generation in response to uncaging of caged inositol-1,4,5-trisphosphate (IP3), so the inhibitory ethanol effect is not due to interference with IP3 receptor function, but must be the result of the demonstrated inhibition of IP3 formation.2+ signaling has previously been shown to be an important factor in controlling the output of glucose from the liver.In this issue of 2+.2+ signal generation, whereas the opposite would appear to be true for the liver .None declared.


The small leucine-rich proteoglycan (SLRP) family is widely expressed in extracellular matrix and aggravates tumor progression. However, epiphycan (EPYC), as a member of the SLRPs family, its biological function in cancer has not been confirmed. Thus, we aimed to clarify the role of EPYC in progression of ovarian cancer (OC), and further analyze the molecular mechanisms implicated in tumorigenesis. Here, we analyzed the differential expression genes of GSE38734, including 4 matched primary OC and metastatic tissues. We obtained OC RNAseqs data from the Cancer Genome Atlas (TCGA) and analyzed the correlation between EPYC expression and OC staging, pathological grading, etc. The expression of EPYC in OC and normal ovarian tissues was compared in Oncomine website. We used siRNAs to interfere the expression of EPYC in ovarian cancer cell line SKOV3. Scratch test, transwell-matrigel chamber, CCK8 assay were used to detect the changes of SKOV3 migration, invasion and proliferation ability after EPYC was interfered. We used R software to make GO and KEGG analysis of related genes of EPYC. We used the Hitpredict website to predict interacting proteins. The results showed that the expression of EPYC in metastatic ovarian cancer was higher than primary ovarian cancer, and that in primary cancer was higher than normal ovaries. After siRNA interferes with EPYC expression, the migration, invasion and proliferation of SKOV3 cells were weakened. EPYC mainly played a role in ECM organization, and involved in PI3K/Akt, focal adhesion signaling pathways. EPYC might interact with PLCG2 and CRK, and be involved in signal transduction. Ovarian cancer (OC) is one of the three major malignant tumors of female reproductive system, more than 90% of which is epithelial ovarian cancer, and its mortality rate ranks first among the three major gynecological malignancies . AlthougSmall leucine-rich repeat proteoglycan (SLRP) family is widely present in the extracellular matrix (ECM), participating in matrix formation, and potentially regulate cancer cell proliferation, angiogenesis and migration . There aThus, in the present study, we compared the expression of EPYC in metastatic OC and primary OC by analyzing expression profiles of GSE38734 . Then, whttps://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE38734. Differential expression genes of GSE38734 were analyzed by R software using limma package. We downloaded the raw data of TCGA database from UCSC Xena (https://xena.ucsc.edu/). LinkedOmics is a publicly available platform for analyzing multiomics data from TCGA database (http://www.linkedomics.org/login.php). We searched for interacted proteins of EPYC on website of HitPredict (http://www.hitpredict.org/). Genes that correlated with EPYC in OCs were obtained from this website, then the corresponding genes were subjected to Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway by R software using clusterProfiler package, as well as the GSEA analysis. Genes correlated with EPYC was analyzed by R software using Pearson correlation analysis.The differential genes expressed in ovarian cancer and metastatic ovarian cancer were downloaded from GEO database  was supplemented in the culture medium. EPYC-siRNAs, as well as the corresponding negative control, were synthesized from RibBio . siRNA transfection were performed using the matching siRNA transfection kit from RibBio .SNAI2, CDH1 was calculated by the method of 2−ΔΔCt with ACTB gene expression as a control. The primer sequences of EPYC, SNAI2, CDH1 and ACTB were shown below  was used to extract the total RNA from cells. According to the protocol, total RNA was reversely transcribed into cDNA using the two-step reverse transcription reagents . SYBR Green Master (ROX) (Bimake.com) was used for qRT-PCR according to the protocol. The relative gene expression of EPYC, wn below Table 1.RIPA lysis buffer  was used to extract total protein of cells. BCA assay kit  was used for protein quantification. Then, equal amount of proteins were loaded for SDS-PAGE electrophoresis. After transferring to PVDF membranes, the membranes were blocked with 5% skim milk for 1.5 hours. After milk blocking, the membrans were incubated with EPYC antibody or β-actin antibody at 4°C overnight . After incubating with horseradish peroxidase (HRP)-conjugated secondary antibody  for 2 hours, protein bands were visualized using the enhanced chemiluminescence detection kit  under the Chemiluminescence system.1*10^5 cells/well were seeded into 24-well plates and incubated for 12 hours to make cells adhere to the wall. Then EPYC-siRNAs and negative control were added into culture medium according to the transfection protocol. After interfering for 24 hours, we used a 200 ul pipette tip to make a “cross-shaped” wounds. After gently washing 3 times with PBS, cells were cultured in RIPM 1640 medium containing 2% FBS for another 24 hours and images were captured under a phase-control microscope.SKOV3 cells were seeded and transfected as described above. 24 hours later, the transfected cells were resuspended in serum-free RIPM1640 medium and added to the upper chambers of the transwell chamber . Add RIPM1640 containing 10% FBS to the lower chamber to induce cell invasion. After culturing for 24 hours, the bottom of upper chambers were fixed by 4% paraformaldehyde for 30 minutes and then stained with 0.5% crystal violet for 30 minutes, and the cells on the upper surface of the membrane were removed by a cotton swab. After washing and the chamber is air-dried, the membranes of the bottom of upper chamber were captured under a microscope for analysis.Cell counting kit-8 (CCK8)  was applied in detecting proliferation of SKOV3 cells after transfecting EPYC-siRNAs and negative control for 0, 24, 48, 72 hours. We operated according to the instructions.t-test was utilized to compare the difference between two independent samples. The log-rank test was used for Kaplan–Meier survival analysis. P < 0.05 was considered statistically significant.Statistical analysis was performed using R software. 2-3 experiments were repeated in our study. Data was presented as mean ± standard deviation (SD), and two-tailed To identify the differential expressed genes in metastatic OCs, we analyzed the expression profiles of GSE38734. The datasets contained the mRNA sequencing of 4 primary OCs and their metastatic OC tissues. Differentially expressed genes (DEGs) were analyzed by using R software, and found out that EPYC was the most elevated gene in metastatic OC  (https://www.proteinatlas.org/). From the HPA website, we learned that EPYC protein was barely expressed in normal ovarian tissue (0/3), but could be moderately expressed in a third of OC tissues . All three endometroid OCs were negative (The human protein atlas (HPA) is a Swedish-based program initiated in 2003 for mapping all the human proteins in cells, tissues and organs  analysis. Gene enrichment analysis showed that EPYC might activate EMT, TGF-β and PI3K/AKT signals pathways , ECM components  and some exosomes , establivia inhibition of miRNA-21. Then, PDCD4 suppressed anti-inflammatory mediators such as IL-10, which creates a proinflammatory tumor microenvironment, thus retarding tumor growth  . SLRPs ar growth , 41. In r growth .In the present study, we found that EPYC was the most overexpressed gene in metastatic ovarian cancer tissues than ovarian cancer by analyzing the GSE38734 raw data. Meanwhile, the expression of EPYC in OC was higher than that in normal ovaries. These results indicated that EPYC may play an important role in the development of OC, which was further confirmed in a subsequent functional experiment in OC cells. Similar to other SLRP members, EPYC has a high correlation with the expression levels of MMPs, CXCLs, and TGF-β . What’s EPYC is a relatively new member of SLRPs. EPYC may affect the biological behavior of OC through a variety of ways, and play an important role in the progression of OC. It is a potential candidate for diagnosis and treatment of ovarian cancer. In future studies, we will further explore and verify the mechanism involved in the action of EPYC.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/LD wrote the manuscript, conducted experiments, and analyzed data. DW analyzed results, and designed the manuscript structure. SC was responsible for public data analysis. WH and RZ modified the manuscript. All authors contributed to the article and approved the submitted version.This study was funded by National Natural Science Foundation of China (grant number: 81902642) and Basic Research on Hygiene Application in Suzhou City (grant number: SYS2019099).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Enzymes catalyzing the hydrolysis of the N-glycosidic bond in nucleosides and other ribosides  with diverse substrate specificities are found in all kingdoms of life. While the overall NH fold is highly conserved, limited substitutions and insertions can account for differences in substrate selection, catalytic efficiency, and distinct structural features. The NH structural module is also employed in monomeric proteins devoid of enzymatic activity with different physiological roles. The homo-oligomeric quaternary structure of active NHs parallels the different catalytic strategies used by each isozyme, while providing a buttressing effect to maintain the active site geometry and allow the conformational changes required for catalysis. The unique features of the NH catalytic strategy and structure make these proteins attractive targets for diverse therapeutic goals in different diseases. The free nicotinamide/nicotinic acid riboside is abundant in breast milk and also originates as a cofactor breakdown product, and together with the free base, is the preferred form of the NAD+ precursor for uptake by cells . A . A R, 3Risozymes . These cisozymes . Indeed,activity .2+-interacting portion mediated by a methylene bridge to the N4’ atom [Alternatives to immucillin-like structures showing strong selectivity toward protozoan NHs have been pursued through structure-based drug design approaches. A series of N-arylmethyl compounds  were synN4’ atom ,89,90. SN4’ atom . Albeit N4’ atom , require2+ ion by the ribose-like moiety of the active site ligands is crucial. Indeed, a comparison of the high-resolution structures of the IAG-NH from T. b. brucei bound to Immucillin-H (0.7 nM Ki) and the UAMC-00363 showed that the only appreciable difference in enzyme-inhibitor contacts is the five-membered ring [T. b. brucei and T. vivax IAG-NHs may result in an up to 10-fold difference in Ki values, thus underscoring the need to validate each inhibitor panel with the actual target from the pathogen.The crystal structures of trypanosomal IAG-NHs provided several clues as to the inhibitor determinants that are responsible for the high affinity. The optimal coordination of the active site Cared ring . The strT. b. brucei IAG-NH was also inhibited by metalorganic compounds, such as a Ni2+·Tris complex formed at a basic pH [2+ is coordinated by the Tris molecule and ordered solvent, and only residue Asp14 of the enzyme directly interacts with the ion. The hydroxyls of the Tris molecule partly mimic the ribose O3’ and O5’ groups, underscoring the importance of these subsites for active-site ligation. It is noteworthy that the transition metal ion can closely approach (3.8 Å) the active site Ca2+, implying that the alkali earth ion charge is substantially shielded by the coordination of the active site aspartates. Although yet unexploited, these findings clearly show that alternatives to ribosyl-containing compounds could be engineered as IAG-NH inhibitors to overcome issues of bioavailability and cross-reactivity with mammalian NPs. Crystallographic fragment-screening approaches may provide further clues as to the process of antiprotozoan drug design.The basic pH . The Ni22+, Mn2+, Co2+, Zn2+, and Ni2+ are non-competitive inhibitors of the enzyme, Cu2+ ions being the most potent with Kii = 0.9 µM. The cocrystal structure of IAG-NH with Ni2+ ions revealed that the ion binds at the monomer-monomer interface, interacting with two histidine residues from opposing subunits. The coordinated ion locks the catalytic loops in a semi-open conformation, preventing the closure of the active-site cavity and the positioning of key catalytic residues [Another approach to enzymatic inhibition could be to target regions other than the active site by changing the equilibrium between active and inactive conformations, favoring the latter through non-competitive inhibition. IAG-NH is an enzyme with half of the sites’ reactivity and requires cooperation of both subunits for efficient catalysis. Divalent transition metal ions such as Curesidues . Thus, tT. vaginalis are both purine and pyrimidine auxotrophic, and the dependence of their life cycle on the genome-encoded NHs could be inferred [+ and NADPH, and further suggests that the enzyme may have a specialized—yet, so far not fully identified—role in different organisms [E. coli YeiK/RihB but also from the differences in the substrate preference of isozymes within the same organism.Although NHs are extremely attractive drug targets due to their absence in mammalian hosts, it remains to be seen whether these enzymes may play a crucial role in the metabolic pathways of other pathogens. Pathogens such as inferred ,93,94. Trganisms . Indeed,Leishmania genus also encode for two NHs in their genome, and their expression has been demonstrated in several isolates of L. infantum and L. donovanii [Leishmania mastigotes’ replication in vitro and in vivo. Moreover, the isozyme NH36 of L. donovanii, the causative agent of visceral leishmaniasis, was found to be one of the most immunogenic proteins from mastigote extracts, and the recombinant NH36 protein (part of the Leishmune© vaccine) is effective at eliciting a curative and protective immune response against murine and canine diseases. Three NH36 subsequences  were used to define minimal epitopes retaining adequate immunogenic properties. Interestingly, the C-terminal (termed F3) region expressed in E. coli co-administrated with saponin elicited a ~36% increase in the protective response against L. chagasi, mostly mediated by IFN-γ/IL-10 CD4+ T cells [As is the case for other protozoan parasites, members of the onovanii ,95,96. T T cells . AlthougGene-directed enzyme prodrug activation therapy (GDEPT) is a therapeutic approach relying on the enzymatic conversion of a non-toxic prodrug to its active form by the product of an exogenous gene expressed in the target cells, typically of neoplastic nature. Severely limited by the targeting of the gene to the tumor cells and the transduction efficiency, GDEPT can greatly benefit from the bystander effect where dying cells transfer the active compound via either passive diffusion or through tight junctions.Although NHs have clearly evolved to catalyze reactions on naturally-occurring nucleoside substrates , their aT. vivax is active on nucleoside substrates such as 6-methylpurine riboside, the hydrolytic product of which, 6-methylpurine, is toxic as it interferes with protein and RNA synthesis, as well as adenine salvage. An innovative liposome-based delivery system has been developed, where the IAG-NH enzyme is encapsulated in liposomes with the bacterial OmpF transporter embedded [The IAG-NH enzyme from embedded . The enzembedded . In bothE. coli has a high catalytic efficiency toward 5-fluoruridine [E. coli PNP was delivered using attenuated Salmonella typhimurium to cancer cells to activate 6-methylpurine 2’-deoxyriboside [The pyrimidine-preferring RihB/YeiK NH from ruridine , and theruridine . Two sitriboside ,101. HenThe quaternary structure of NHs may prove extremely relevant in the design of prodrug activators. Indeed, the construction of chimeric enzymes catalyzing subsequent reactions required for activation of the toxic drug and thus improved efficacy has been tested in several cases. The product of the hydrolytic reaction catalyzed by NHs is not toxic to cells per se, but rather requires further condensation with PRPP through the action of PRTases, to yield the mononucleotide that will eventually either be incorporated in RNA molecules, or after reduction to the corresponding nucleoside, will interfere with thymidine synthesis or DNA replication. Thus, a chimeric NH-PRTase enzyme would encode for the two crucial activities that yield a mononucleotide, for instance, 5-FUMP, which cannot escape the cell and will lead to antitumoral cytotoxicity. PRTases are known to oligomerize to quaternary structures that range from dimers to tetramers ,103, andInitially considered redundant or exclusive to nucleobase-auxotrophic parasites, NHs are now recognized as widespread and possessing extremely diversified functions in different organisms. The field will largely benefit from close-knit interaction between structural studies, molecular analysis of mutants, and validation of high-throughput genetic analysis to ascertain the role of the enzymes and related proteins in each organism. The catalytic versatility of NHs warrants further exploration of their therapeutic potential as prodrug activators, and they remain attractive targets for development of drugs against parasitic diseases.