In addition, TLR9 was associated with better disease-free survival (Log-rank p < 0.05). In the multivariate Cox proportional-hazards model for OS, the TLR/TLR9/GATA3 score was found to be an independent prognostic factor for OS .Biliary tract cancer (BTC) refers to a heterogenous group of epithelial malignancies arising along the biliary tree. The highly aggressive nature combined with its silent presentation contribute to the dismal prognosis of this tumor. Tumor-infiltrating immune cells (TIICs) are frequently present in BTC and there is growing evidence regarding their role as therapeutic targets. In this study, we analyzed the immune cell infiltration in BTC and developed a promising immune signature score to predict prognosis in BTC. Immunohistochemistry (IHC) was carried out on tissue microarray sections from 45 patients with resectable cholangiocarcinoma for the detection of 6-sulfoLacNAc+ monocytes (slanMo), BDCA-2+ plasmacytoid dendritic cells (pDC), CD8+ or CD4+T-lymphocytes, CD103+ cells, GATA3+ cells, Toll-like receptor (TLR) 3, 7 and 9-expressing cells as well as programmed cell death protein 1 and programmed cell death ligand 1 positive cells. Data from the IHC staining were analyzed and correlated with clinicopathological and survival data. High expression of TLR7, TLR9, and GATA3 was associated with improved overall survival (OS, Log-rank  Biliary tract cancer (BTC) comprises a diverse cluster of malignancies that can arise anywhere along the biliary tree, including the gallbladder . This highly heterogeneous group of tumors occurs after a malignant transformation of various cells lining the biliary tract . BTC is In Western countries, BTC occurs in 1–2 cases per 100,000 people, whereas in some Eastern countries the incidence is as high as 85 per 100,000 people . This diThese cancers have a dismal prognosis due to their aggressiveness and late diagnosis which severely compromises the effectiveness of available therapeutic options . SurgicaThe prognosis of BTC after surgical resection is still burdened by a relapse rate ranging from 50% to 70% ,20. Due Patients who underwent surgical resection of a biliary tract cancer at the Department of Surgery, Bonn University Hospital, between 2013 and 2017 were included in this study. Patients’ demographic data including gender, age, as well as tumor and treatment-related data were collected. Survival data were retrieved from the patients’ records. Overall survival (OS) was calculated from the date of surgery to the last follow-up or death. Disease-free survival (DFS) was calculated from the date of surgery to the date of tumor relapse or last follow-up. Pathological characteristics of the tumors were obtained from the clinical records. All tumors were thoroughly restaged according to the most recent TNM classification, 8th Edition . The usa® Adhesion Microscope Slides, . The TMA sections were deparaffinized with xylene  and rehydrated using decreasing concentrations of graded ethanol  to water . For slan monocytes (slanMo) and pDC stainings, antigen retrieval was achieved by boiling the slides in citrate buffer  at pH 6.0 for 20 min. The tissue samples were then stained overnight at 4 °C with either the polyclonal goat anti-BDCA-2 antibody  to evaluate pDCs or the monoclonal mouse anti-slan antibody DD2  to analyze slanMo. Then, tissues used for pDC staining were incubated with a mouse anti-goat antibody solution  for 60 min. Afterward, all tissues were incubated with dextran-labeled antibodies against mouse immunoglobulins  for 30 min. pDCs and slanMo were visualized by the alkaline phosphatase-based EnVisionTM detection system according to the manufacturer´s instructions (Dako).A tissue microarray (TMA) was constructed manually. Standard hematoxylin and eosin stained 3 µm slides were obtained to identify regions with 100% tumor tissue. For each sample, four to six representative tumor cores of 1 mm diameter were transferred from original FFPE blocks to the TMA blocks. Immunohistochemistry (IHC) was carried out on TMA sections according to standardized protocols. Antibodies to 6-sulfo LacNAc (slan), BDCA-2, CD8, CD4, CD103, GATA3, Toll-like receptor 3 (TLR3), TLR7, TLR9, PD-1 and PD-L1 were used. The details of antibodies and dilutions used are summarized in For CD4  and CD8 antibodies , antigen retrieval was achieved by boiling the slides at 99 °C in citrate buffer at pH 6.0 for 20 min. IHC was performed using the semi-automated platform Autostainer 480 S . All supplementary reagents were provided by Medac.®, Cambridge, UK), GATA3 , TLR3 , TLR7 , TLR9 , PD-L1  and PD-1  the antigen retrieval was performed by boiling the slides at 99 °C at pH 8.0 for 20 min. IHC was conducted using semi-automated platforms . All supplementary reagents were provided by Ventana Medical Systems, Inc., Tucson, AZ, USA. Antibodies details are summarized in For CD103  with an algorithm for IHC staining. The slides were digitalized with a Zeiss Mirax scanner  and saved in MIRAX-format. All whole slide digital images were assessed for scanning artifacts. Mean values were calculated for each case. The median was used as cut-off for dichotomization in “high CD4+/CD8+” and “low CD4+/CD8+” groups. Regarding PD-1 and PD-L1 staining, tumors with clusters of positive cells (>1%) were classified as “high PD-1/PD-L1”, whereas tumors without clusters of positive cells were classified as “low PD-1/PD-L1”. Tumor samples with infiltrating slan+ cells were classified as “slanMo+” and tumor samples without positive cells were considered as “slanMo-”. CD103 and BDCA-2 staining was evaluated by means of the percentage of positive cells in a TMA core and samples where then classified as “high/low CD103/ BDCA-2”. Mean values were calculated for each tumor. For GATA3 quantification, positively stained cells were counted for each core and the mean value was calculated for each case. Tumor samples were then dichotomized in “high GATA3” or “low GATA3” staining according to the median values. For TLR3 and TLR9 staining, tumor cells positivity was evaluated. Samples with moderate or high positive tumor cells were classified as “high TLR3” or “high TLR9”, respectively. For TLR7, samples with TLR7+ infiltrating immune cells were classified as “high TLR7”. Samples with only faintly positive cells or a negative reaction were classified as “low TLR3”, “low TLR7” or “low TLR9”.p < 0.05 was considered statistically significant.Statistical Analysis was performed with SPSS Statistics Version 22 , and in the R environment  . The prip < 0.05) were added to the multivariate model for OS. A multivariate Cox proportional-hazards model for DFS was then applied. The IHC biomarker with prognostic value for DFS (TLR9) was used as the only marker score for the multivariate Cox proportional-hazards model for DFS. All clinical factors that were prognostic when considered alone (p < 0.05) were added to the multivariate model for DFS.The IHC biomarkers with the best prognostic value for OS  were combined in an immune marker score. The score was calculated as follows: cases with negative IHC staining for TLR7, with a low TLR9 and low GATA3 positivity were classified as negative (score 0). Samples with positive IHC staining forTLR7, or TLR9 high positivity and a GATA3 positivity were classified as low positive with a score of 1 (two positive markers). Samples with a positivity for TLR7, high positivity for TLR9 and GATA3 were classified as high positive and a score of 2 was given to these samples (three positive markers). Three patients were excluded from the analysis due to missing markers due to tissue fragmentation. A multivariate Cox proportional-hazards model for OS was then used to evaluate the prognostic value of the immune marker prognostic score adjusted for significant prognostic clinical factors. All clinical factors that were prognostic when considered alone (n = 17). pBTC was diagnosed in 11 patients (24%). dBTC and GBC were diagnosed in 27% (n = 12) and 11% (n = 5) of the patients, respectively. Adjuvant chemotherapy was administered postoperatively in 58% of the patients (n = 26) . A positive nodal status (N1) was found in 53% (n = 24). Four patients had peritoneal carcinosis, which was detected after pathological examination . Most of the tumors were moderately differentiated , and the majority did not present lymphatic invasion . Vascular invasion was present in 16% of the lesions (n = 7). In 53%, a perineurial invasion was detected (n = 24). Eleven patients showed microscopically positive margins after resection. The clinicopathological characteristics of the cohort are summarized in Samples from 45 patients  were analyzed. The most represented entity was iBTC  . Most of2 (range 17–4314 cells/mm2) whereas median CD8+ cell density was 125 cells/mm2 (range 2–2216 cells/mm2). Median percentage of CD103+ cells was 5% (range 0–26%). In the majority of the samples  an infiltration of Slan-Mo was found. Median percentage of positive BCDA+ cells was 1% (range 0–9%). In 7% of the probes (n = 3), clusters of PD-1+ lymphocytes were detected. In about half of the samples , clusters of PD-L1+ cells were observed. Most of the samples were classified as “high TLR3” , whereas 53% (n = 24) and 91% (n = 39) of the samples were classified as “high TLR7” and “high TLR9”, respectively. Representative examples of immunohistochemistry staining are displayed in Median CD4+ cell density was 652 cells/mmn = 10, 83% vs. n = 4, 23% vs. n = 5, 45%; p < 0.05). Interestingly, in all samples from patients with GBC, a high expression of CD103 was observed, and the difference in expression pattern was significant compared to dBTC . The percentage of samples with high TLR7 expression was significantly higher in dBTC compared to pBTC and GBC . For slan, CD4, CD8, TLR9, PD-1, PD-L1, and GATA3 no difference regarding expression and tumor localization was observed . In the group with high TLR9 expression, the median OS was 33.2 months (95% CI 17.6–49.0 months), whereas the group with low TLR9 expression displayed an OS of 8.3 months . Regarding GATA3 expression, median survival was not reached by the high expression group, whereas in the low expression group, the median OS was 12.9 months . Low expression/negativity of TLR9 was also associated with shorter DFS. In the group with high expression of TLR9, median DFS was 24.9 months (95% CI 6.1–43.7 months), and 4.4 months (95% CI 1.4–7.5 months), in the low-expression group (log-rank p = 0.007).  in the group with high TLR7 expression versus 20.1 months (95% CI 7.5–32.6 months) in the low TLR7 expression group (log-rank  0.007). .p = 0.020). A similar yet not statistically significant trend was found between low TLR9 expression vs. high TLR9 expression . A high GATA3 expression was also significantly correlated with a better prognosis . In addition, the univariate analysis showed that Stage III tumors were significantly associated with worse OS when compared to stage I tumors   .p = 0.013). Among the clinicopathological parameters, a positive R status and a positive vascular invasion were significantly associated with worse DFS   .A score was calculated using the three markers with the best prognostic significance for overall survival , as described in the Methods section. Due to tissue fragmentation in at least one IHC staining, a score could not be calculated for three samples. The patients were then excluded from the analysis. Clinicopathological characteristics of the patients based on the score subgroups are summarized in p < 0.01). The median DFS of the “score 2” group was higher than in the “score 0” group . The median DFS in the “score 1” group was 35.0 months (CI 95% 2.5–27.5 months), which was found to be significantly longer when compared to the DFS in the “score 0” group (log-rank p = 0.006), and similar to the DFS in the “score 2” group (log-rank p = 0.644). , and 7 months (95% CI 0.0–14.9 months) in the group with a score of 0 (log-rank  0.644). .p = 0.003; “score 1” vs. “score 2”: HR 4.45, 95% CI 0.95–20.80, p = 0.058). In the univariate analysis for DFS, TLR9 was the only marker significantly associated with DFS and therefore the only one added to the multivariate Cox proportional-hazards model for DFS. In the multivariate analysis, TLR9 was found to be an independent prognostic factor for DFS . R status and vascular invasion were also found to be independent prognostic factors for disease-free survival   that areVarious studies have underlined the antitumoral function of TLR9 in cancer. The anti-tumor effect of TLR9 signals is derived from the enhanced secretion of type-1 IFN, including IFNa from pDCs through TLR9 activation . HoweverGATA3 belongs to a family of tissue-specific transcription factors regulating multiple developmental pathways . In T ceOur study has some limitations. First, our monocentric cohort was too small to build a validation cohort. A multicentric study with larger cohorts could address this limitation in the future. In addition, this study implied some intrinsic limitations due to its retrospective design and the results must be interpreted accordingly. Based on our findings, we introduce a novel IHC-based score that could help to stratify patients with BTC. Moreover, we demonstrated that a TLR9 signature could help to identify patients with higher risk of recurrence after resection for BTC.


Swine reproduction efficiency is determined by the fertility potential of the sow and sperm quality. The objective of this study is to compare boar sperm motility and kinematic features to evaluate their relationships with reproductive success after artificial insemination (AI). In this study, the movement patterns of boar ejaculates were analyzed by a computer-assisted semen analysis (CASA)-Mot system, and the kinematic values of ejaculate clusters were assessed. The semen of the Pietrain boars showed more linear trajectory of the spermatozoa, while curvilinear velocity and oscillatory movement characterized the semen of the Duroc × Pietrain boars. The offspring of sows inseminated with Pietrain boars showed significantly lower number of stillbirths. In addition, ejaculate grouping into clusters did not have a predictive capacity on litter size variables. Nevertheless, the kinematic variables of the ejaculate may have a predictive, albeit reduced, capacity regarding litter size variables. The results of this study therefore open up possibilities for future assessments of fertility.®v1 CASA-Mot system for eight kinematic parameters. Ejaculate clusters were characterized using multivariate procedures, such as principal factors (PFs) analysis and clustering methods (the k-means model). Four different ejaculate clusters were identified from two kinematic PFs which involved linear trajectory and velocity. There were differences (p < 0.05) between the sperm kinematic variables by sire line. There was no statistical difference (p > 0.05) between dam lines and ejaculate clusters in fertility variables. The discriminant ability of the different kinematics of sperm variables to predict litter size fertility was analyzed using receiver operating characteristics (ROC) curve analysis. Curvilinear velocity (VCL), average path velocity (VAP), amplitude of lateral head displacement (ALH), and beat-cross frequency (BCF) showed significant, albeit limited, predictive capacity for litter size fertility variables . The kinematic analysis of the ejaculates in clusters did not have a predictive capacity for litter size variables.The aim was to determine the relationship between kinematic parameters of boar spermatozoa and fertility rates of sow, as well as to assess the effect of sperm clusters on the fertility capacity of the ejaculate. Semen samples were collected from 11 sexually mature boars. Samples were analyzed by an ISAS Several countries have implemented frequent artificial insemination (AI) in pigs ,2,3, whiThe sperm quality needs to be evaluated to understand the fertility and genetic value of the boar. Because of this, boars are housed in an artificial insemination station to optimize management, health, and fertility ,13. CompMotility evaluation is based on kinematic patterns ,17. SperSeveral authors have indicated that the ejaculate is composed of a heterogeneous population of spermatozoa according to their kinematic variables ,32,33,34The principal parameters to determine sow fertility are litter size (LS), piglets born alive ,43, stilThe experiment was conducted at a commercial swine farm  in 2019–2020 in the northwest of Costa Rica , following the laws and regulations for experiments on live animals in Costa Rica. This study was performed following ethical principles, and with the approval of the Committee of Centro de Investigación y Desarrollo de la Agricultura Sostenible para el Trópico Húmedo at the Costa Rica Institute of Technology (CIDASTH-ITCR), according to Section 08/2020, article 1.0, DAGSC-100-2020.n = 8) and Pietrain boars (n = 3)), 23.3 ± 8.5 months of age at the beginning of the experiment and with known fertility, were used as semen donors in this study. For the study, breeding boars were housed individually in well-ventilated pens with average temperature of 25.60 ± 2.94 °C during the time of the experiment. Ejaculates were collected in rainy season. Females came from four crossbred dam genetic lines (DL: York (Y), Landrace (L) and Pietrain (P); with the following crossing schemes YLP-50 (¼ Y × ¼ L × ½ P), YLP-75 (1/8 Y × 1/8 L × 3/4 P), YLP-87.5 (1/16 Y × 1/16 L × 7/8 P); and Y-L-50 (½ Y × ½ L)). All the females were bred at the farm and they came from within maternal crossing schemes such as the continuous 3-generation cross between YLP hybrid sows and P boars. Mean sow parity for all dam genetic lines was 4.10 ± 2.76. The animals were fed with the standard breeder mixture (made on the farm), containing maize, soybean meal, mineral mixture, and common salt, to fulfill their nutrient requirements [Eleven sexually mature and healthy boars from two commercial terminal sire lines . Total piglets born per litter (TPB), piglets born alive (PBA), stillbirth (SB), number of mummies (MP), and litter weight  were used as fertility variables, and those parameters were measured at the farrowing time.9 spermatozoa. From each boar, 3.64 ± 0.81 ejaculates were obtained. Samples from each ejaculate were evaluated for motility, and only ejaculates with at least 70% motile spermatozoa and 85% morphologically normal spermatozoa were used. The concentration was measured with Spermacue  following established protocols [® tube  and maintained at 37 °C for 30 min before use.Semen samples were collected in the morning, once per week, using the “gloved-hand” technique  and immerotocols . Samples® D4C20 disposable counting chambers  were used after being pre-warmed at 37 °C. After thorough mixing of the diluted semen samples, a volume of 2.7 µL was distributed along the counting chamber race by capillarity to fill it completely. Analyses were conducted using the CASA-Mot system ISAS®v1  fitted with a video-camera , with 25 frames acquired per field at a frame rate of 50 Hz and final resolution of 768 × 576 pixels. The camera was attached to a microscope UB203 (UOP/Proiser R+D) with a 1× eyepiece and a 10× negative phase contrast objective (AN 0.25), and an integrated heated stage maintained at a constant temperature of 37.0 ± 0.5 °C. The CASA settings used were a particle area between 10 and 80 μm2 and connectivity of 11 μm. The percentage of total motile cells and progressive motility (%) corresponded to spermatozoa swimming forward quickly in a straight line. The following parameters defined progressive motility: straightness  ≥45% and average path velocity (VAP) ≥25 µm·s−1, defined as the average velocity over the smoothed cell path. A single technician carried out the assessments of sperm morphology. Sperm were classified as having normal or abnormal morphologic features following WHO strict criteria [For the analysis of motility, ISAScriteria . A total−1), corresponding to the straight line from the beginning to the end of the track; curvilinear velocity , measured over the actual point-to-point track followed by the cell; average path velocity , the average velocity over the smoothed cell path; amplitude of lateral head displacement , defined as the maximum of the measured width of the head oscillation as the sperm swims; beat-cross frequency , defined as the frequency with which the actual track crosses the smoothed track in either direction; motility (%), defined as the percentage of total motile cells; and progressive motility (%), corresponding to spermatozoa swimming rapidly forward in a straight line. Three progression ratios, expressed as percentages, were calculated from the velocity measurements described above: linearity of forward progression (LIN = VSL/VCL·100), straightness (STR = VSL/VAP·100), and wobble (WOB = VAP/VCL·100).The CASA analyses were performed in seven microscope fields on a total of at least 600 cells per sample. The CASA-Mot variables assessed in this study included straight-line velocity  was performed on these data to derive a small number of linear combinations that still retained as much information as possible from the original variables. Prior communalities for this analysis were estimated from the maximum absolute correlation coefficient between each variable and any other. The number of principal factors (PF) to be extracted was determined from the Kaiser criterion, namely by selecting only those with an eigenvalue >1. The KMO (Kaiser-Meyer-Olkin) statistic was also obtained  as a mea2. Second, the optimal number of clusters obtained in the previous analysis was used as the target number of clusters in a non-hierarchical K-means cluster analysis [Further, an analysis was conducted to classify the ejaculates into a reduced number of clusters, based on scores obtained from factor analysis. This was accomplished in two phases, combining hierarchical and non-hierarchical clustering procedures. First, factor scores for all ejaculates were clustered hierarchically using the Ward Minimum Variance method . From thanalysis .p < 0.05. Further, pairwise comparison between cluster means were performed by the Tukey-Kramer test. Results were presented as mean ± standard deviation of the mean. All data were analyzed using the SAS 9.4 [ANOVA was further applied to evaluate statistical differences between clusters for all kinematic variables. The threshold for significance was defined as  SAS 9.4  statistiSow fertility variables were analyzed using the Generalized Linear Mixed Models (GLMM). The response variables were litter weight, litter size, piglets born alive, stillbirth, and number of mummies. A normal distribution with an identity link function was assumed for litter weight, while a Poisson distribution with a log identity link function was assumed for all other response variables. Ejaculate clusters, obtained from multivariate analysis, was considered as the main fixed independent factor in the model. Other fixed factors with potential effects on sow fertility were also added to the model, such as dam line, sire line, dam × sire line interaction, nested boar within sire line, month of farrowing, pregnancy length, different parities, and length of period between previous and present ejaculate. A random residual effect was also added to the model to account for correlations between different ejaculates obtained from the same boar. GLMM analysis was performed with the SAS 9.4  statistiThe diagnostic test with a dichotomous outcome (positive/negative fertility test results) of the different kinematic semen variables to predict litter size fertility was analyzed using receiver operating characteristic (ROC) curve analysis. The approach of diagnostic test evaluation uses sensitivity and specificity as measures of accuracy of the test, in comparison with standard status (farrowing). The sensitivity (true positive rate) and specificity (true negative rate) of each kinematic variable vary across the different thresholds, and the sensitivity was inversely related with specificity. The plot of sensitivity versus 1-Specifity is called receiver operating characteristic (ROC) curve and the area under the curve (AUC). AUC varies from 0.5 (test with no discriminatory ability) to 1 (perfect discriminatory ability). An ROC was also used to calculate the elective breaking point  for each kinematic sperm variable. The analysis may also be used to determine the optimal cut-off value .6/mL, 231.98 ± 63.08 mL and 82.04 ± 23.73 × 109, respectively. The sperm concentration (million/mL) was 378.63 ± 134.98 in the Duroc × Pietrain crossbred and 361.00 ± 112.35 in the Pietrain. Total motility (%) of boar samples was 77.36 ± 11.17, with an overall range of 35.05–93.69%. The progressive motility of sperm (%) was 63.76 ± 11.96. Average total motility (%) for Duroc × Pietrain and Pietrain boars was 81.28 ± 7.76 and 65.61 ± 11.73, respectively (p < 0.05). The progressive motility (%) was 67.00 ± 10.05 (Duroc × Pietrain) and 54.04 ± 12.19 (Pietrain) (p < 0.05).Sperm concentration, volume of semen, and total spermatozoa in the ejaculate were 374.23 ± 129.24 × 10Principal factors analysis indicated a KMO statistic of 0.56, and final communality estimates were above 0.85 for all kinematic variables, except BCF (0.05). According to Kaiser criterion, two significant PF can be extracted from these data, both accounting for 98% of the total variance. The first PF, defined as linear trajectory (PF1), was responsible for 53% of the variance and was mainly associated with the kinematic variables LIN, STR, WOB, and VSL, with the largest correlation being for LIN (0.99). The second PF, defined as velocity (PF2), was strongly associated with the variables VCL, VAP, VSL, and ALH, with the largest correlation being for VCL (0.98) . This fa2 = 0.75). The model was adjusted in the cluster procedures analysis with R2 in each repetition for better validation . These ejaculates present a spermatozoa with highest BCF and ALH . Cluster 2 (EC2) included sperm characterized by high VSL (42.91 ± 2.76 µm·s−1), and the highest values of LIN and STR . Cluster 3 (EC3) contained the ejaculates whose spermatozoa had a high oscillatory movement, indicated by WOB and BCF , and an intermediate value of STR (75.72 ± 5.58%). Cluster 4 (EC4) exhibited ejaculates whose sperm were less linear and progressive, as indicated by the lowest VSL and VAP , together with the lowest values of LIN and STR .Boar ejaculates were grouped into four clusters according to hierarchical Ward’s minimum variance, followed by non-hierarchical k-means clustering procedures . The kinp < 0.05) between the sperm kinematic variables by sire line. The kinematic variables of semen of the Pietrain boars showed more linear trajectories of the spermatozoa, whereas semen from Duroc × Pietrain boars were characterized by curvilinear velocity and oscillatory movement of the sperm (p < 0.05) between ejaculate clusters for total piglets born per litter (TPB), stillbirth (SB), and litter weight . There were no significant differences between ejaculate clusters (p > 0.05) for piglets born alive (PBA) and number of mummies (MP). However, some trends were observed. EC3 had higher PBA (10.25 ± 1.32), but higher MP (0.32 ± 0.17); moreover, LW was also higher (18.83 ± 1.71 kg). EC2 was characterized by intermedium fertility rates in all categories, exhibiting a higher value in PBA (9.55 ± 1.28) than EC1 and EC4. Finally, EC4 had the lightest litter (11.47 ± 2.03 kg); because of this, EC4 presents the lowest TPB (7.64 ± 1.13), and also reported the lowest SB (0.12 ± 0.07) and intermedium MP (0.19 ± 0.14). The fertility variables characterized according to ECs indicate that EC3 showed the highest litter size, while EC4 presented the lowest value of total piglets born per litter. EC2 and EC3 presented higher values of stillbirth than EC1 and EC4 (p > 0.05), except for significantly fewer stillbirths in Pietrain boars (The mean fertility rate was 69.60 ± 21.67%. There were no differences between sire lines for this variable. There were differences (he sperm . Fertili and EC4 . The ferin boars .p > 0.05) between dam lines. The hybrid (Y-L-50) presents the highest value in TPB (10.44 ± 0.96), PBA (9.45 ± 0.92), and SB (0.65 ± 0.21). YLP-87.5 showed the lowest MP (0.13 ± 0.08), followed by Y-L-50 (0.15 ± 0.11). The heaviest litter was for YLP-75 (15.53 ± 1.32 kg), which represents 103.22% of the value of Y-L-50 (p > 0.05). Pietrain had lower values of MP (0.13 ± 0.09), but Duroc × Pietrain presented a higher value of TPB (10.27 ± 0.65) and PBA (9.08 ± 0.61) (Fertility parameters did not show significant differences (f Y-L-50 . The Pie ± 0.61) .−1 VCL, 42.40 µm·s−1 VAP, and 2.59 µm ALH. Similarly, ejaculate clusters showed limited predictive capacity for litter size variables (data not shown).The sperm kinematic variables with significant results in the ROC curve analysis are presented in 9 spermatozoa per AI dose. Our multivariate analysis found four ejaculate clusters, where EC1 was characterized by rapid and progressive spermatozoa; this cluster had only ejaculates from Duroc × Pietrain boars. Moreover, EC4 was mainly ejaculates provided by Duroc × Pietrain boars, and was characterized by slow and non-progressive sperm. The major proportion of Pietrain ejaculates was in EC3; this cluster had ejaculates with slow and progressive spermatozoa. In EC2, both sire lines were better distributed, and the ejaculates were characterized by medium velocity and linear progressively motile spermatozoa.The number of sows inseminated by a single ejaculate depends on the concentration and quality of the semen . This qu9 spermatozoa per AI dose, no relationship with fertility parameters is found [p > 0.05) on the fertility data. Non-relevance of the difference between boar racial groups could be due to the fact that the clusters are very close in Euclidean distance with respect to the centroids, and some ejaculates were assigned to a specific group (cluster) but maintained some motile spermatozoa and/or kinetic patterns similar to ejaculates from another cluster. The main obstacle to estimating the fertility of the boar is that it is necessary for a large number of sows to be inseminated over a long period of time, requiring an extended time period for the study, and during this time the boar fertility could change [p > 0.05) between dam lines in the fertility variables, even though those values showed highly variable percentages compared to Y-L-50. However, we suggest that there is biological and economic importance because of the genetic improvement achieved by selecting sows and boars to increase the number of live born piglets and the survival proportion [p < 0.05 from each cluster of boar ejaculates, and in some cases indicated that differences between cluster could be considered irrelevant. Other studies carried out in other species, such as cattle, have described results that indicate that the differences between sperm subpopulations are not relevant [Some authors have found that sperm parameters, such as motile spermatozoa, do not predict litter size ,65. Otheis found ,67. Thisis found ,43,48, pis found ,50, and is found ,50. Our d change ,68. The d change ,69,70,71d change , housingd change , nutritid change , duratiod change , endocrid change , and sowd change . In the d change ,77,78. Id change ,80,81. Td change . There woportion . The preoportion , and thioportion , which ioportion . To explrelevant . On the We have shown that kinematic analysis of boar ejaculates reveals kinematically separate populations. There were differences between the sperm kinematic variables by sire line. However, there was no overall significant difference between dam lines assessed by multivariate procedures. The fertility variables characterized according to the sire genetic line did not show differences, except for significantly fewer stillbirths in Pietrain boars. Sperm kinematic variables may have a predictive capacity for litter size variables, albeit a limited one. Nevertheless, the analysis of the ejaculates into clusters did not have a predictive capacity for litter size variables.


Background: Polydactyly is a prevalent digit abnormality characterized by having extra digits/toes. Mutations in eleven known genes have been associated to cause nonsyndromic polydactyly: GLI3, GLI1, ZRS regulating LMBR1, IQCE, ZNF141, PITX1, MIPOL1, FAM92A, STKLD1, KIAA0825, and DACH1.Method: A single affected family member (IV-4) was subjected to whole-exome sequencing (WES) to identify the causal gene. Bi-directional Sanger sequencing was performed to segregate the identified variant within the family. In silico analysis was performed to investigate the effect of the variant on DNA binding properties.Results: whole-exome sequencing identified a bi-allelic missense variant  in exon nine of GLI1 gene located on chromosome 12q13.3. With the use of Sanger sequencing, the identified variant segregated perfectly with the disease phenotype. Furthermore, in silico analysis of this DNA binding protein revealed that the variant weakened the DNA binding interaction, resulting in indecorous GLI1 function.Conclusion: Herein, we report a novel variant in GLI1 gene, causing autosomal recessive post-axial polydactyly type A (PAPA) type 8. This confirms the critical role of GLI1 in digit development and might help in genotype–phenotype correlation in the future. Polydactyly is also known as hexadactyly or polydactylism, which occurs due to extra complete on incomplete fingers or toes. It is one of the most common limb deformities observed prenatally or instantly after birth. The incidence of polydactyly is approximately 0.3–3.6/1,000 observed in live births and 1.6–10.7/1,000 in the general population . In addiBroadly polydactyly is classified as a syndromic form that is associated with other phenotypes such as Bardet–Biedl syndrome (BBS), Ellis–Van Creveld (EVC) syndrome, split hand/foot malformation (SHFM), and syndactyly, while in nonsyndromic polydactyly, it is inherited as an isolated entity. Until now, more than 300 syndromic forms of polydactyly have been characterized .Moreover, polydactyly is further classified into three main types according to the severity of the duplication defect and position, viz., pre-axial polydactyly (PPD), where the additional digit is attached to the thumb or the big toe, or post-axial polydactyly (PAP), where the additional digit is located on the fifth finger. Furthermore, PAP is subclassified into post-axial type A with a fully developed digit and post-axial type B, where the extra digit is developed as a skin tag or rudimentary. Both type A and type B polydactyly may differ in penetrance, severity, and inheritance pattern . The thiGLI3, GLI1, ZRS regulating LMBR1, IQCE, ZNF141, PITX1, MIPOL1, FAM92A, STKLD1, KIAA0825, and DACH1) and three other unknown loci have been mapped on different chromosome 13q21–32, 13q13.3–21.2, and 19p13.1–13.2, which have been linked with isolated polydactyly , zinc finger protein 141 gene , GLI family zinc finger 1 , IQ Motif Containing E , KIAA0825 , family with sequence similarity 92 member A1 , and Dachshund Homolog 1   , 2021 (T 603803) .GLI1 protein consists of 1,106 amino acids and acts as a mediator of hedgehog signaling pathway. GLI1 protein becomes active after proper binding of hedgehog molecule to its receptor, which ultimately results in transcription and further translation of target genes, which are associated with the limb development and patterning of digits ; GLI1 gene. In addition to this, mutant modeling of this DNA binding protein was performed, which revealed a decline in the DNA binding activity of the mutant protein that ultimately affects the functional property of GLI1 protein.Our study recruited a consanguineous family segregating PAP type A in an autosomal recessive manner. Our finding revealed a novel homozygous missense variant by whole-exome sequencing (WES) followed by Sanger sequencing in The institutional review board (IRB) of King Abdullah International Medical Research Center (KAIMRC), Riyadh, Saudi Arabia, approved this study. Written informed consent/approval forms were signed by elderly family members for conducting the research experiments and thereafter publication of all clinical data/results in the journals and conferences including radiographs and photographs of affected patients recruited in this study.In our present study, a single family was recruited in which three members of the fourth generation  were exhibiting nonsyndromic PAP type A. A pedigree was drawn after communication with the most experienced and older family members . The detThe affected individuals were clinically evaluated in a local government hospital. All the affected individuals had classical PAP type A phenotypes. The age of the affected individuals  at the time of examination was between 25 and 35 years. All the affected individuals were born normally at full-term pregnancies without any additional complications. The affected individual (IV-4) showed bilateral PAP in both hands and feet . RadiogrApproximately 3–5 ml of the peripheral venous blood samples was collected from the affected individuals  and unaffected individuals  in a cell-free DNA blood collection EDTA tubes ; and each tube was labeled with a unique medical record number (MRN) and accession number.For each blood sample, 2 ml of the buffy coat was collected after spinning whole blood at 4,000 rpm for 3–4 min. From each buffy coat sample (2 ml), genomic DNA extraction experiments were performed by using QIAamp DNA Blood Midi Kit from Qiagen , according to the manufacturer’s protocol. The quantification of extracted DNA was determined by Qubit™ dsDNA HS Assay Kit (Cat. No. Q32851), as densitometric methods are not specific for DNA.DNA of a single affected individual (IV-4) was exome sequenced using the Ion S5™/Ion S5™ XL Sequencers or the Ion Proton™ Sequencer , and libraries were prepared using the Ion AmpliSeq™ Exome RDY Kits (Cat. No. A38262). WES was performed in the following steps, as per manufacturer’s protocol.For amplification, 56 µl of gDNA (100 ng) was taken and mixed with 14 µl of 5× Ion AmpliSeq™ HiFi Mix, and a total of 70 µl was made by adding nuclease-free water (NFW). Thereafter, the master mix was dispensed in a single row (12 Column) in the 96-well plate. The plate was transferred to the thermal cycler, and the cycling condition was 99°C for 2 min (1 cycle) followed by 99°C for 15 s and 60°C for 16 min (10 cycles).After completion of the amplification reaction, the 12 targets were combined into a single well no. 6, and thereafter, 6 µl of FuPa reagent was added to the combined target amplification reaction to bring the total volume up to 66 µl. Finally, the plate was transferred to the thermal cycler, and the program was set as 50°C, 55°C, and 60°C each for 20 min.After completion of partial digestion, 6 µl of diluted Ion Xpress™ Barcode Adapters was mixed, then 12 µl of switch solution was added, and finally at last 6 µl of DNA ligase was added. Finally, after completion of adaptor ligation, the library was purified by using 80 µl of AMPure™ XP reagent and 150 µl of freshly prepared 70% ethanol as per company protocol .® Quantitation Kit (Cat. No. 4468802) as per instruction given in the manufacturer’s protocol. The library was diluted to make the final concentration to ∼100 pM. The Ion Chef™ reagent was kept outside at room temperature for 45 min before loading of libraries. The other consumables and solution cartridge were placed at an appropriate position into the Ion Chef™ equipment  (The quantification of the library was carried out by using the Ion AmpliSeq™ library by qPCR with the Ion Library TaqMan4484177) . Finally4484177) .The Ion S5™ ExT Sequencing Reagents cartridge was kept for 45 min to equilibrate the reagent at room temperature. After that, Sequencing Reagents cartridge, Ion S5™ ExT Wash Solution bottle, and Ion S5™ Cleaning Solution bottle were kept at an appropriate position in the Ion S5™ instrument . Finally, Ion 540™ chip (Cat. No. A34292) was used to perform initialization. After the completion of initialization, the chip was immediately transferred to the Ion S5™ instrument, and a sequencing run was performed .https://basespace.illumina.com). Variant filtration steps also focused on the autosomal recessive mode of inheritance given priority as per the pedigree analysis. In addition to that, homozygous and heterozygous variants as well as pathogenic to likely pathogenic variants were also given priority during filtration steps.After completion of the sequencing run, the data were generated into a BAM file, which is automatically converted into a Variant Calling File (VCF) by the instrument, and the VCF was uploaded into a Base Space Illumina hub (https://varsome.com/), MutationTaster (http://www.mutationtaster.org/), FATHMM (http://fathmm.biocompute.org.uk/), and DANN (https://cbcl.ics.uci.edu/) were used to determine the pathogenicity of the identified variant in our study. The frequency of the identified variant in the general population was calculated by using available online databases including Exome Variant Server (EVS), 1,000 Genomes, ExAC, gnomAD, and/or in-house 2000 exome database.Furthermore, different online software such as Varsome  using standard methods .https://www.rcsb.org/) with PDB Id, 2GLI. The S247L (Ser to Leu mutation at 247 position) mutant was generated through Pymol v.4.6.0The three-dimensional crystal structure of five-finger GLI1, a DNA binding protein in complex with DNA, was retrieved from Protein Data Bank (PDB) (GLI1 gene (GLI1 gene causing PAP type A (PAPA) type 8 (PAPA8) as listed in Human Gene Mutation database (HGMD) , as described previously ). The outcomes of WES data analysis and the filtration steps resulted in the identification of a novel homozygous missense mutation  located on chromosome 12q13.3 (GRCh37) in exon nine of LI1 gene . Until ne (HGMD) .The bi-directional Sanger sequencing experiments were performed for affected and unaffected individuals by using exon specific primers (forward: 5′-CCTGGATTTATGGGACCTCA-3′) and (reverse: 5′-GAATGGATGGGTTGGGAAGTA-3′), which showed correct segregation of a novel homozygous missense mutation (c.1010C > T) in the affected individuals, viz., IV-2, IV-4, and IV-5; heterozygous carrier in the individuals, viz., III-2 and III-3; and homozygous wild type in the individuals, viz., IV-1 and IV-3 .Pathogenicity of the novel missense variant  and its deleterious effects on the structure and function of protein was confirmed by using several available online software .GLI1 variant was screened in different online databases such as gnomAD (http://gnomad.broadinstitute.org/), ExAC (http://exac.broadinstitute.org), dbSNP and the 1,000 Genomes Project , or in-house 2000 exome database (https://www.ncbi.nlm.nih.gov/projects/SNP/). The identified novel variant was not detected in the homozygous state as mentioned in online tools. Until now, almost six different variants have already been reported for GLI1 gene for PAPA8 as listed in HGMD  controls the number of digits of the autopod during early embryonic development by regulating the activity of GLI1, GLI2, and GLI3 . GLI proGLI1 gene have been associated with polydactyly PAPA8 and PPD type I ; GLI1 gene (NM_001167609.1) located on chromosome 12q13.3 in a consanguineous Pakistani family exhibiting hallmark features of PAPA. The variant (p(Ser337Leu)]) is considered disease causing by several online bioinformatics tools (GLI1 (GLI Family Zinc Finger 1) is a member of the Kruppel family of zinc finger proteins. Variants in cs tools  and is hcs tools . HoweverThe highly conserved polar Serine amino acid at position 337 is substituted by a non-polar aliphatic leucine amino acid. This substitution, signposted by the 3D structural analysis, might decline the DNA binding. Thus, alteration in the protein’s function might lead to abnormal digit features in the affected individuals .GLI1 gene. PPD features were not observed in patients reported here. Isolated PAPA features observed in the present family were a mirror to the features reported previously [In the present investigation, bilateral PAP in both hands and feet with complete development of extra digit was observed in all affected members of the family . Palencieviously . FeatureGLI1 gene have been associated with isolated PAP A/B . Our data further support the evidence that biallelic variants in GLI1 cause recessive PAP in humans. Identifying variants in recently known polydactyly genes provides clinicians, genetic counselors, and researchers valuable clinical information. These might help in genetic counseling of the affected family and prevent such disorders in upcoming generations.In conclusion, we detected a novel missense variant in


Magnetosomes are membrane-enclosed iron oxide crystals biosynthesized by magnetotactic bacteria. As the biomineralization of bacterial magnetosomes can be genetically controlled, they have become promising nanomaterials for bionanotechnological applications. In the present paper, we explore a novel application of magnetosomes as nanotool for manipulating axonal outgrowth via stretch-growth (SG). SG refers to the process of stimulation of axonal outgrowth through the application of mechanical forces. Thanks to their superior magnetic properties, magnetosomes have been used to magnetize mouse hippocampal neurons in order to stretch axons under the application of magnetic fields. We found that magnetosomes are avidly internalized by cells. They adhere to the cell membrane, are quickly internalized, and slowly degrade after a few days from the internalization process. Our data show that bacterial magnetosomes are more efficient than synthetic iron oxide nanoparticles in stimulating axonal outgrowth via SG. Mechanical stretch can modulate several different cellular functions, such as the electrical activity of cardiac muscle , osteogennection ,6. Internnection . MNPs arnnection , with a nnection . Static nnection  that allMagnetospirillum gryphiswaldense, they consist of a cuboctahedral core of chemically pure magnetite (Fe3O4) that is naturally enveloped by a biological membrane [Magnetosomes are organelles that enable magnetotactic bacteria (MTB) to align along geomagnetic field lines (magnetotaxis) . In the membrane ,10. The membrane ,12,13,14membrane . Precisemembrane ,17,18,19Magnetosomes can be isolated from the cells and purified with the intact membrane, thereby ensuring that the particles are colloidal stable and dispersible ,14. In aM. gryphiswaldense were shown to possess distinctive abilities to induce stretch-growth of mouse hippocampal neurons. Thereby, a strong cellular interaction was observed even at very low concentrations.In our study, magnetosomes isolated from −1), cell viability was only slightly affected, even for more sensitive primary cells [The cytotoxicity/biocompatibility of bacterial magnetosomes has been tested on various mammalian cell lines, including primary cells as well as cancer cells ,43,44,45ry cells ,37.M. gryphiswaldense were isolated and purified with their intact biological membrane (2 kg−1) [−1)  ,47. Magn−1) [−1) C require−1 magnetosomes, 2+ and Fe3+ that is mark of particle dissolution. Collected data point that magnetosomes are mainly intact at 1 dpt, as documented by the brown-stained pellet and the low intracellular level of Fe ions  enables them to be stretched through the dragging force created by an external magnetic field ,6. In th−1). Each experiment was repeated four times and analyzed by two different operators. In each experiment and with each dose tested, we found a statistically highly significant increase (p < 0.0001) in axonal length in comparison to the control groups .The elongation analysis was carried out on cells labelled 4 h after plating with magnetosomes and exposed from day in vitro (DIV) 1 to DIV3 to the magnetic field  or a null magnetic field (labelled as control group). l groups B,C. To dlculated D. In lin2kg−1), i.e., the most powerful inducer of stretch-growth that have been tested so far from our team [−1, using respectively, 3.6 µg Fe mL−1 of SPIONs, and 1.25 and 2.5 µg Fe mL−1 of magnetosomes. Surprisingly, the treatment with 2.5 µg Fe mL−1 of magnetosomes provided a statistically significant increase in the length of neurites compared to samples treated with SPIONs 3.6 µg Fe mL−1 (p = 0.021). We were wondering if this increase could be associated to differences in the cell–particles interactions between the two groups. The study of the axonal ultrastructure shows that magnetosomes adhere to the cell membrane of developing axons and, after internalization, localize as single particles within the axon , magnetic properties, and surface coatings. Here, for comparison, we selected the commercially available SPIONs  and of the local Ethical Committee of University of Pisa, in conformity with the Directive 2010/63/EU (project license approved by the MoH on 22 November 2018). C57BL/6J mice  were used. They were kept in a regulated environment  with a 12 h light-dark cycle with food and water ad libitum.−1  in DPBS ; chemical digestion and mechanical dissociation were performed to isolate hippocampal neurons. Cells were seeded in high-glucose DMEM  with 10% fetal bovine serum , 100 IU mL−1 penicillin , 100 μg mL−1 streptomycin , and 2 mM GlutaMAX  on poly-D-lysine -coated surfaces at a density of 160 cells mm−2. Cells were incubated at 37 °C in a saturated humidity atmosphere containing 95% air and 5% CO2. After 4 h, the medium was replaced with Neurobasal-A medium  modified with B27 , 2 mm GlutaMAX, 50 IU mL−1 penicillin, and 50 μg mL−1 streptomycin.The dissection of both hippocampi of P0–P1 mice of both sexes was conducted in a solution of D-glucose 6.5 mg mL2kg−1, as stated from the supplier. They have an organic shell preventing aggregation, made of glucuronic acid, and the hydrodynamic diameter is 100 nm.SPIONs, commercially available , have an inorganic core of iron oxide, ~75 ± 10 nm in diameter, and saturation magnetization of 59 AmM. gryphiswaldense MSR-1 [3, 0.74 mM KH2PO4, 0.6 mM MgSO4, 50 μM Fe3+-citrate, 3 g L−1 soy peptone, and 0.1 g L−1 yeast extract, at pH 7.0) as previously described [g, 4 °C, 20 min). The cell pellets were subsequently washed with 20 mM HEPES and 5 mM ethylenediaminetetraacetate (EDTA), at pH 7.2, and stored at −20 °C until further use.The wild-type (WT) strain of se MSR-1 ,49 was gescribed . Cells wChemicals were purchased from Merck  or Carl Roth  with purity grades ≥98%.M. gryphiswaldense were resuspended in 50 mM HEPES, 1 mM EDTA, at pH 7.2, and cell disruption was performed by passing the suspension 3–5 times through a microfluidizer system  equipped with a H10Z interaction chamber at 124 MPa. Afterwards, the crude extract was passed through a MACS magnetic-separation column  placed between two neodymium-iron-boron magnets (each 1.3 T). Thereby, the magnetosomes were retained within the column, whereas non-magnetic cellular compounds passed the column and were instantly eluted. The column was subsequently washed with 50 mL 10 mM HEPES, 1 mM EDTA, at pH 7.2, followed by 50 mL high-salt buffer  to remove electrostatically bound impurities, and again by 50 mL 10 mM HEPES and 1 mM EDTA, at pH 7.2. The magnets were finally removed, and the magnetosomes were eluted with ddH2O. As further purification step, the particle suspension was centrifuged through a 60% w/v sucrose cushion  for 2 h at 200,000× g and 4 °C. During centrifugation, the magnetosomes formed a pellet at the bottom of the tube (due to their high density), whereas residual impurities were retained in the cushion. Lastly, the particles were resuspended in 10 mM HEPES, at pH 7.0, and stored in Hungate tubes at 4 °C under a nitrogen atmosphere.Magnetosomes were isolated and purified as previously described ,14. Cell2O and analyzed using an Analytik Jena contrAA 300 high-resolution atomic absorption spectrometer  equipped with a 300 W xenon short-arc lamp  as continuum radiation source. The equipment presented a compact high-resolution double monochromator (consisting of a prism pre-monochromator and an echelle grating monochromator) and a charge-coupled device (CCD) array detector with a resolution of about 2 pm per pixel in the far ultraviolet range. Measurements were performed at a wavelength of 248.3 nm using an oxidizing air/acetylene flame. The number of pixels of the array detector used for detection was 3 . All measurements were carried out in quintuplicates (n = 5), each as a mean of three technical replicates.The iron content of suspension of isolated magnetosomes was determined by atomic absorption spectroscopy (AAS). For this, 25–50 µL of magnetosome suspension was mixed with 69% nitric acid  and incubated for 3 h at 98 °C. Samples were subsequently filled up to a volume of 3 mL with ddHMagnetosomes were fluorescent-labelled by reaction with DyLight 488 Amine-Reactive Dye . Thereby, the labelled particles were shown to exhibit an up to 12-fold increased fluorescence compared to EGFP magnetosomes  .−1 stock solution in dimethylformamide) was added to 1 µg magnetosomes  and incubated in the dark for 2 h at 16 °C. Excess dye was removed by extensive washing in which the particles were several times pelleted by centrifugation  and, after discarding the supernatant, resuspended in 10 mM HEPES, at pH 7.0. Success of the labelling reaction was confirmed by fluorescence measurements (535 nm) using an Infinite M200pro plate reader .Briefly, 1.5 µg of the fluorescent dye . The particles were collected by low-spin centrifugation  and resuspended in a small volume of ddH2O. After determination of the Fe content by AAS, the suspensions were diluted to a final Fe concentration of 500 µg mL−1. For application under sterile conditions, magnetosomes were sterile filtrated as described previously . SuspensM. gryphiswaldense cells and isolated magnetosomes, specimens were deposited onto carbon-coated copper-mesh grids . Isolated particles were additionally negatively stained with 2% uranyl acetate. TEM was performed on a JEOL 1400  with an acceleration voltage of 80 kV. Images were taken with a Gatan Erlangshen ES500W CCD camera.For transmission electron microscopy (TEM) analyses of 4, 1% K3Fe(CN)6, and 0.1 M sodium cacodylate buffer), stained with our homemade staining solution [For localization studies, neurons, both control and stretched, were fixed with 1.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, at pH 7.4. After rinses with the same buffer, cells were post-fixed in reduced osmium tetroxide solution  were cut with a UC7 LEICA ultramicrotome  and collected on 300 mesh copper grids .Images for morphological characterization have been collected with a Transmission electron microscope Zeiss LIBRA 120 Plus operating at 120 KeV equipped with an in-column omega filter.mag) of M. gryphiswaldense cultures were monitored by photometric measurements at 565 nm as reported previously [565 directly correlates with the cellular growth. Cmag represents a light-scattering based proxy for the average magnetic orientation of bacterial cells and relates to the average magnetosome numbers in cell populations, allowing semiquantitative estimations of the particle content.The optical density (OD) and magnetic response . Measurements were performed in the automatic mode (wavelength 638 nm) at 25 °C on highly diluted particle suspensions using DTS1070 cuvettes.The summary of the main properties of bacterial magnetosomes and a comparison with the commercial SPIONs used in this work is provided in −1 in the radial centrifugal direction [Experiments were conducted in 35 mm Petri dishes placed inside a Halbach-like cylinder magnetic applicator, which provided a constant magnetic field gradient of 46.5 Tmirection .For stretching assay 80,000 cells were seeded on a 14 mm glass coverslip. Alternatively, seeding of hippocampal neurons was performed in a 2-well silicone insert , at a density of 100,000 cells per well. After letting the neurons attach for 4 h, the silicone insert was removed in order to generate a cell-free gap region of 500 µm. For both supports used, particles were added 4 h after seeding (DIV0.17). At DIV1, the culture was placed in the magnetic applicator (Stretch group) or outside (Control group). At DIV3, samples were fixed and stained for immunofluorescence.Toxicity of magnetosomes was evaluated by performing dose–response assays. At DIV3, cells were fixed and stained for immunofluorescence and the number of viable neurons was counted.After lysis, cell membranes were precipitated from the lysate by centrifugation . An iron assay kit  was performed on cell lysate for intracellular iron quantification, and the absorbance was measured at a wavelength of 590 nm.An iron stain kit  was applied for particle localization. In addition, fluorescent magnetosomes were used. For the immunostaining performed for stretching and toxicity analysis, samples were fixed in 2% PFA, permeabilized with 0.5% Triton X-100, and blocked with 5% FBS at room temperature. Primary antibody TUBB3  was diluted in 3% FBS/0.2% Triton X-100 in DPBS and incubated overnight at 4 °C. Samples were then washed and incubated with secondary antibody  and Hoechst 33342 . All images were acquired using a fluorescent microscope .For the immunostaining performed on 2-well silicone insert, cells were fixed in 2% PFA and 7.5% sucrose, permeabilize in 0.1% Triton X-100 and blocked with 5% goat serum (GS) at room temperature. Primary antibodies TUBB3  and KDEL  were incubated overnight at 4 °C. Samples were then washed and incubated with secondary antibody  Hoechst 33342 . All images were acquired using a laser scanning confocal microscope .l was evaluated using the plugin NeuronJ [The analysis of the elongation was performed using image analysis software ImageJ. Neurite length  NeuronJ , and 120l has been considered, and the thickness s was calculated as s = A/l, A being the axon area related to that path. Area was calculated from images after threshold normalization, binary conversion.For axon thickness, a population of 40 axons was analyzed from 10× magnification images (randomly acquired). For each axon, the longest path p ≤ 0.05. Statistical power analyses have been performed with G-power software.Data were plotted and analyzed with GraphPad software, version 6.0. Significance was set at Neurons are mechanosensitive cells, and the application of extremely low force positively modulate axonal elongation, neurite branching, and neuron maturation ,63,64,65


Sea salt dominated wet deposition volume-weighted concentrations year-round without much variability in its mass fraction in contrast to stronger seasonal changes in PM2.5 composition where fresh sea salt was far less influential. The highest mean annual deposition fluxes were attributed to Cl−, NO3−, SO42−, and Na+ between April and October. Nitrate is strongly correlated with dust constituents  in precipitation samples, indicative of efficient partitioning to dust. Interrelationships between precipitation chemistry and aerosol species based on long-term surface data provide insight into aerosol–cloud–precipitation interactions.This study focuses on the long-term aerosol and precipitation chemistry measurements from colocated monitoring sites in Southern Florida between 2013 and 2018. A positive matrix factorization (PMF) model identified six potential emission sources impacting the study area. The PMF model solution yielded the following source concentration profiles: (i) combustion; (ii) fresh sea salt; (iii) aged sea salt; (iv) secondary sulfate; (v) shipping emissions; and (vi) dust. Based on these results, concentration-weighted trajectory maps were developed to identify sources contributing to the PMF factors. Monthly mean precipitation pH values ranged from 4.98 to 5.58, being positively related to crustal species and negatively related to SO These iAlthough few studies focusing on ACI over the WNAO have primarily relied on ship and/or aircraft data . Even thFor studies relating aerosol and precipitation chemistry, it helps to study locations with broad ranges in both aerosol and meteorological conditions. In this regard, coastal areas influenced by both natural and anthropogenic emissions are fitting as they are exposed to a wide assortment of aerosol types. On the western edge of the WNAO, Southern Florida fits into this category as it experiences influence from local and regional sources that are both biogenic and anthropogenic in nature , in addiAdditionally, smaller bodies of water  can create circulations similar to sea breeze leading to conditions to form thunderstorms. Intercomparison of aerosol and wet deposition chemical data for Southern Florida can suggest what aerosol types participate in CCN/IN activation and what the resultant composition is of the wet deposition derived from clouds. Wet deposition flux magnitudes of different species are relevant for understanding impacts on surface ecosystems, which was already an active research area decades ago in the study region as part of the Florida Atmospheric Mercury Study ,22.2.5; (iii) precipitation chemistry profile; and (iv) interrelationships between precipitation and aerosol chemistry.This work aims to report on long-term aerosol and precipitation chemistry measurements from colocated monitoring sites at the Everglades National Park (NP) in Southern Florida. The analysis combined field measurement data, transport modeling, and source apportionment modeling to characterize the temporal trends and interrelationships between aerosol and precipitation chemistry from 2013 to 2018. The results and discussion are structured as follows: (i) summary of meteorological profile; (ii) sources of PM2.2.1.The Everglades NP hosts the precipitation monitoring station ) managed by the National Atmospheric Deposition Program/National Trends Network (NADP/NTN) and the Environmental Protection Agency (EPA) Interagency Monitoring of Protected Visual Environments (IMPROVE) aerosol monitoring station . Both sites are located approximately 25 km from the coast. The Everglades NP is a unique ecosystem formed by subtropical wetlands . Located2.2.Established in 1977 by the U.S. State Agricultural Experiment Stations (SAES), the NADP/NTN (hereafter referred to as NADP) began monitoring wet deposition across the U.S. to measure its effects on the environment. The network continued to expand throughout the years and currently has over 260 sites with the Wisconsin State Laboratory of Hygiene at the University of Wisconsin-Madison as the NADP Program Office. In 1987, the IMPROVE program was set up with 30 monitoring sites located in national parks as part of the visibility monitoring network , which c4+), calcium (Ca2+), chloride (Cl−), magnesium (Mg2+), nitrate (NO3−), potassium (K+), sodium (Na+), and sulfate (SO42−)). Precipitation-weighted concentrations were calculated and used in this study [The NADP site collected weekly precipitation samples for pH, conductance, and water-soluble ions concentrations (ammonium (NHis study .2.5): water-soluble ions , elements , arsenic (As), bromine (Br), calcium (Ca), chromium (Cr), cupper (Cu), iron (Fe), lead (Pb), Na, magnesium (Mg), manganese (Mn), nickel (Ni), phosphorus (P), potassium (K), rubidium (Rb), selenium (Se), silicon (Si), sodium (Na), strontium (Sr), titanium (Ti), vanadium (V), zinc (Zn), and zirconium (Zr)), three fractions of elemental carbon , and four fractions of organic carbon . This study also used mass concentration data for fine soil and PM with diameters less than or equal to 10 μm (PM10). The difference between PM10 and PM2.5 is referred to as coarse particulate matter (PMcoarse).At the IMPROVE site, 24-h filter samples were collected every third day . Relevan2.5 fraction were measured by ion chromatography, while the elemental mass fraction was measured by either X-ray fluorescence or particle-induced X-ray emission. The seven carbon fractions mentioned above were measured using the IMPROVE’s thermal optical reflectance (TOR) method [The water-soluble ions in the PM) method ,36. The ) method . The EC ) method . The cha) method . Additio) method  and Solo) method . Monthly2.3.Meteorological data at the Everglades NP site (25.3900° N 80.6800° W) for precipitation accumulation and temperature (minimum and maximum) were obtained from the NADP site. Wind speed and average temperature were obtained from the EPA air quality system (AQS) database . Planeta2.4.2.4.1.2.5 [total = 658) to determine sources and evaluate corresponding contributions impacting the sampling site. The PMF model was not applied to the precipitation chemistry data as the NADP dataset had fewer species , reducing the number of possible sources that can be identified; keeping the native precipitation species rather than PMF source factors allows for better intercomparison with other studies reporting the same water-soluble ion concentrations. Thirty-two species  were included in the analysis. Each species was categorized depending on the signal to noise ratio (S/N). Species were classified as “Bad” if the S/N ratio was less than 0.5, “Weak” if the S/N ratio was greater than 0.5 but less than 1, and “Strong” if the value was greater than 1 [2.5 on the model’s results, it was added as a “Total Variable” and categorized as a “weak” species [Q values while running DISP and BS-DISP tests. Finally, results were evaluated for the maximum change in the sum-of-squares (dQmax). A dQmax value of 4 was chosen as it provides robust model outcomes with the smallest error [Positive matrix factorization (PMF) modeling was carried out using the U.S. EPA PMF version 5A. Positive matrix factorization has been widely implemented to conduct source apportionment of PM2.5 –46. The r than 1 . Species species . Based o species , missingst error .2.4.2.2.5 sources  model was used to determine the spatial distribution and transport pathways of potential PMs . This as  combustion; (ii) fresh sea salt; (iii) aged sea salt; (iv) secondary sulfate; (v) shipping emissions; and (vi) dust. The mass concentration of different species and the percent of their mass accounted for by a specific source are shown in 3.2.1.2.5. Dominant constituents influenced by this source factor include the following, with percent values being the fraction of that constituent accounted for by this source: OC3 (69.7%), P (64.4%), EC1 (60.9%), OC4 (60.2%), Cu (59.6%), Zn (56.7%), OC2 (50.5%), Br (44.9%), EC2 (43.6%), As (41.8%), Pb (38.7%), NO3− (23.7%), and K (22.5%). There were more minor contributions from this source to Ca (15.4%), Cr (12.3%), Sr (7.7%), Mn (7.5%), Ni (4.8%), Fe (4.6%), Cl (3.7%), V (3.1%), Ti (3.0%), Al (2.1%), Si (1.4%), and Mg (0.1%).The first factor, defined as combustion, was identified as having influence from biomass burning, vehicle emissions, industrial activities, and fuel/oil sources. Combustion accounts for 17.0% of the total PM3− [Biomass burning has been shown in past work to be an especially large contributor to EC1, P, OC2, OC3, and OC4 , which a3− , while m3− . Zinc an3− . Inciner3−  likely c3− .−3). The relative contribution remained relatively stable throughout the year with an average value of 0.18 ± 0.06 , Na (43.1%), and Mg (39.3%). The main differentiating characteristic between the fresh sea salt and the aged sea salt factor was the replacement of Cl− by NO3− owing to sea salt’s chemical reactivity with acids [3−, and 0% to Cl−. Chloride depletion occurs due to the reaction between sodium chloride (NaCl) and acids such as sulfuric and nitric acids (H2SO4 and HNO3) to form sodium nitrate (NaNO3) and sodium sulfate (Na2SO4) [3– in the supermicrometer size range in contrast to SO42− [The fresh and aged sea salt factors represent 12.6% and 6.6% of the total PMth acids ,73. More(Na2SO4) –77, resp(Na2SO4) –82. Sizeto SO42− –85.−3) with a peak during March revealing a higher contribution (0.30) when compared to the rest of the year (−3), with slightly higher levels between March–May (0.40–0.47 μg m−3). This coincides with the months exhibiting the highest cumulative mass concentrations from three PMF source factors known to emit acids, including combustion, shipping emissions, and secondary SO42−. In contrast to the fresh sea salt, aged sea salt concentrations were located closer to the coast of Florida and over the Caribbean Sea , which is secondarily produced via gas-to-particle conversion processes from its main precursor SO2; however, this factor includes other important species that can be secondarily produced as well, such as secondary organic aerosol (SOA) represented by OC2 (14.2%). This factor also included contributions to Br (41.8%), Pb (33.6%), Zn (32.3%), K (27.0%), EC1 (17.9%), Cr (14.6%), Mn (10.9%), As (7.7%), OC4 (7.5%), Na (7.1%), Sr (6.4%), Mg (4.9%), Al (4.4%), Ca (4.0%), P (3.7%), NO3− (3.4%), Si (3.3%), Ti (2.9%), Fe (2.9%), Ni (2.4%), and Cl (1.0%). As SO42− was most pronounced in this factor, it is worth noting that it can be secondarily produced from ocean emissions of dimethylsulfide (DMS) [2. For instance, Pb and Zn are coemitted with SO42− precursors from municipal solid waste incinerators [42− precursors include: emissions from gasoline vehicles  [The next factor, identified as secondary SOde (DMS) , while anerators  located nerators . Other sand OC2) ,91; Cr eand OC2) ,92; and 2.3%), K .0%, EC1 and OC2) ,93.42− (−3), coincident with reduced precipitation scavenging. Secondary SO42− relies on production via aqueous-phase chemistry , which n factor . The samn factor .3.2.4.2.5. The high contributions to V (73.7%), Ni (67.1%), SO42− (46.9%), EC2 (46.4%), Pb (23.4%), OC2 (21.6%), As (21.3%), EC1 (20.5%), P (18.8%), and OC4 (9.9%) confirmed the primary source of this factor as they are commonly found in low-cost residual oil used as ship fuel [The shipping emissions factor represents 20.2% of the total PMhip fuel –99. The hip fuel  as a mar2.5 monthly profile and fractional contribution for shipping emissions in −3) and September-October (1.20–1.40 μg m−3). Compared to the other five factors, this factor showed the least variability among all the aerosol sources. The shipping emissions WCWT map , Ti (86.9%), Al (85.5%), Fe (83.9%), Mn (75.9%), Cr (67.3%), Sr (47.5%), Ca (42.4%), and K (31.0%) clearly defined this factor as dust [−3 and 0.51 to 0.65, respectively  are shown in 2 . Howeve2  exhibited the highest concentrations of all ions studied. Sea salt particles are an important source of CCN due to their large size and hygroscopicity, which allows for easier droplet activation as compared to other aerosol types [−, SO42−, and Na+ in precipitation as compared to PM2.5 may be partly explained by how the wet deposition data include influence from particles of any size participating in cloud processes or subcloud scavenging. In contrast, the aerosol data were limited to ≤2.5 μm. Sea salt typically ranges from 0.01 and 10 μm [The total concentration sum of the eight speciated ions was elevated between October and April , coincidol types ,124–126.nd 10 μm , with th− and Na+ since sea salt is not expected to show such an increase in a particular month. We also examined the available data back to 1980 and did not observe unusual concentrations as the ones observed during this period. Further analysis of the dataset (2013–2018) showed elevated Cl− and Na+ in mid-October 2015 and 2016  was still close to that of pure seawater . Th. Th− andtrations –S16. Theinterest . Future −1 month−1) was observed during June when precipitation reached its maximum value. The highest mean annual deposition fluxes were attributed to Cl− (0.53 kg ha−1 month−1), NO3− (0.32 kg ha−1 month−1), SO42− (0.32 kg ha−1 month−1), and Na+ (0.30 kg ha−1 month−1). On the other hand, Ca2+, K+, and Mg2+ showed lower deposition fluxes, each being less than 0.1 kg ha−1 month−1. However, deposition fluxes for all three of those ions showed an increase during the summer months compared to other times of the year due most likely to dust influence.In comparison to the precipitation ion concentrations, the opposite monthly trend was observed for the average monthly deposition fluxes, where higher values were observed during the high precipitation months  and followed by Na+ (0.15–0.24), NO3− (0.08–0.23), and SO42− . Gr. Gr− . Therefore, it is an important result that the sea salt impacting precipitation was relatively pristine when considering the various other sources impacting the area.Chloride-to-sodium ratios are commonly used as a marker for the presence of sea salt and possible Clepletion ,136. Marr of 1.8 . The ratr of 1.8 ,139). Fo aerosol . Little ic acids , which wNew York . Strong nd Werby  reported42− and NO3− are suggestive of contributions from anthropogenic emissions and combustion of oil products  [42− mass fractions were highest between March-April and September (0.20–0.22), whereas NO3− fractions were highest in June–July (0.21–0.23). Additional contributions to the SO42− mass fraction may be attributed to sea salt and secondary production from DMS emissions over the ocean. Additionally, another species commonly linked to SO42− and NO3− anions owing to secondary salt formation is NH4+. Ammonium showed a monthly profile (range = 0.03–0.08) more accordant with SO42−, suggestive of potential secondary salt formation between those two species . Mass size distributions of NO3− commonly indicate significant contributions to the coarse aerosol fraction. Thus, its different monthly profile may be linked to a summertime association with coarse dust, unlike secondarily produced salts.High mass fractions for SOtraffic)  due to t2+, Mg2+, and K+) had mass fractions ranging from 0.01 to 0.06 throughout the year. All three species peaked at different times of the year, pointing to different influential sources. For instance, Ca2+ peaks in July-August (0.05) and is thus temporally coincident with the dust season, whereas Mg2+ peaks between October and February (0.03), which overlaps with when the sea salt constituent mass fractions peak. Lastly, K+ peaked in August and September (0.05–0.06), which was likely linked to dust.The least abundant species . The strength of the relationship can be categorized as follows for the absolute r value: r = 0–0.2: weak; r = 0.21–0.4: moderately weak; r = 0.41–0.6: moderate; r = 0.61–0.8: moderately strong; and r ≥ 0.81: strongly correlated [p-value < 0.05), but a few were notable. Species that were positively correlated with pH included Ca2+ , NO3− , NH4+ , and K+ . The three cations were commonly associated with dust and smoke. Precipitation pH showed a moderate weak negative correlation with the acidic species SO42− (r = −0.29), consistent with past work in the study region [42− concentration, reported in March. Even though high concentrations of SO42− were recorded throughout the year, it is possible that these do not have a larger impact on the precipitation pH due to the neutralizing effect of NH4+ [2+ [Another way to examine the precipitation chemistry data is to understand the relationship between the species present in precipitation. rrelated . Interesy region . The low of NH4+ ,146 and NH4+ [2+ .− and both Na+ (r = 1.00) and Mg2+ (r = 0.99), which is expected from a near-coastal location influenced by sea salt [2+ was with NO3− (r = 0.89), which suggests partitioning of NO3− to dust, as shown in previous studies [2+ and SO42− (r = 0.41) suggests that NO3− preferentially participates in reactions with coarse particles that serve as CCN/IN, supported by the monthly profile of mass fractions in the NADP data mentioned earlier. However, there could be potentially enhanced uptake of nitric acid (HNO3) in existing droplets enriched with Ca2+ [3− was weakly correlated with sea salt constituents , which was surprising as many studies associate NO3− with sea salt aerosols [3− available to those salt particles seeding the regional clouds. Certainly, more size-resolved measurements of particles in the region can help solidify some of the speculated conclusions reached here regarding relationships between NO3− with both sea salt and dust particles.The strongest correlation among all of the species was between Clsea salt ,83,148.  studies –151. A mith Ca2+ ,85,152. 3.4.Concentrations of the precipitation species are related to the PMF source factor and aerosol species concentrations. The PMF and aerosol chemistry concentrations were averaged to the time resolution of NADP data to allow for a direct comparison in the form of a correlation matrix . Notable+, Mg2+, and Cl−) were similarly related to the same species in the aerosol samples and the PMF fresh sea salt factor. These results suggest how a directly emitted marine aerosol type similarly impacts both PM2.5 and wet deposition at a coastal site and show that the technique used to relate the aerosol and precipitation data offers promise to build on in future studies. Sulfate from NADP samples showed a weak positive correlation with Na+ (r = 0.20) and Cl− (r = 0.17) in the aerosol dataset (but not Mg2+), which was not surprising as it is an important constituent in natural sea salt. The same sea salt constituents in wet deposition samples interestingly exhibit a weak negative relationship with fine soil in the aerosol dataset. This relationship can likely be related to the seasonal variation of these species. Sodium, Mg2+, and Cl− in the precipitation data peak during the fall months (SON), while aerosol fine soil levels peak during the summer months (JJA). The lack of any relationship between the sea salt constituents in the NADP datasets with the aged sea salt factor from PMF analysis implied that the region’s precipitation was more representative of natural sea salt rather than sea salt that had undergone chemical reactions.First, the major sea salt constituents in wet deposition  between the two datasets, the same applies to SO42− owing to how the secondary SO42− source factor exhibited a moderately weak correlation with precipitation SO42− (r = 0.23). Precipitation SO42− also exhibited moderately weak (r = 0.30) and weak (r = 0.14) correlations with fresh sea salt and combustion PMF factors, respectively. The weak to moderately weak positive correlations for precipitation SO42− can be explained by how SO42− was present in sea salt, and it was also produced from secondary formation from both biogenic [4+ exhibited a weak positive correlation with only the secondary SO42− PMF factor (r = 0.15), consistent with ammonium sulfate formation. Since precipitation NO3− did not exhibit any correlation with the same species in the IMPROVE dataset, this suggests that its involvement with wet deposition was based on processes unrelated to surface NO3− formation in PM2.5, including its association with CCN/IN larger than 2.5 μm [3 partitioning to cloud and precipitation drops aloft, as has been documented in other works [Similar to how sea salt components showed a statistically significant correlation  and PMcoarse (r = 0.18). It was already suggested by 3− preferentially partitions to coarse dust particles , and these results support that notion. The lack of a correlation between precipitation Ca2+ and either fine soil or the dust PMF factor could be due to the latter two IMPROVE parameters being limited to the PM2.5 fraction, whereas the dust particles involved with precipitation processes are larger. An interesting hypothesis motivated by 2+ and NO3− partly because soil dust can be entrained in biomass combustion plumes ..2+ and b2.5. In contrast, larger particles  have been shown here to be especially influential in the collected wet deposition samples. Therefore, any species associated with the coarse sea salt that served as the CCN/IN and any species taken up by existing drops  would not have been similarly observed at the surface IMPROVE monitoring site. As The lack of other relationships in 4.2.5 and wet deposition. The two datasets were intercompared to gain insights into aerosol–precipitation relationships. The points below summarize the findings of this study:2.5 (percentage contributions): (i) secondary sulfate (23.0%); (ii) dust (20.6%); (iii) shipping emissions (20.2%); (iv) combustion (17.0%); (v) fresh sea salt (12.6%); and (vi) aged sea salt (6.6%).The Southern Florida coastal site was impacted by a diverse source of pollutants. The following six sources were identified in decreasing contribution to total PM42−, whereas they were positively related to dust presence as based on the crustal tracer species Ca2+.Monthly mean precipitation pH ranges from 4.98 ± 0.31 (March) to 5.58 ± 0.51 (May). Values of pH were negatively related to the acidic anion SO−, NO3−, SO42−, and Na+ between April and October, coinciding with months experiencing the most precipitation. Although lower in magnitude, enhanced fluxes of Ca2+, K+, and Mg2+ in summertime coincided with the main dust season.The highest mean annual wet deposition fluxes were attributed to Cl2.5 owing partly to how the sea salt particles seeding precipitation drops likely exceed 2.5 μm. Aged sea salt was shown to be far less influential in the region’s precipitation.Fresh sea salt was the dominant component in the region’s precipitation, unlike surface PM2.5, it was much less influential in volume-weighted wet deposition concentrations and mass fractions as compared to sea salt.Even though dust plays a large role in PM2+ and both aerosol NO3− and PMcoarse was linked to NO3− preferentially partitioning to coarse dust.A weak positive association between precipitation Ca42− in the NADP and IMPROVE datasets demonstrated that the combined use of these long-term datasets could be useful for other regions to indirectly examine aerosol-precipitation interactions.Statistically significant correlations  between related parameters indicative of dust, sea salt, and SOThe analysis of long-term precipitation chemistry and aerosol data from a coastal site in the Southeast U.S. was used to gain insight into emissions sources impacting both PMSI


Escherichia coli RNase H as a model, and examined its catalytic mechanism and its substrate recognition modes, using single-molecule FRET. We discovered that RNase H acts as a processive exoribonuclease on the 3′ DNA overhang side but as a distributive non-sequence-specific endonuclease on the 5′ DNA overhang side of RNA:DNA hybrids or on blunt-ended hybrids. The high affinity of previously unidentified double-stranded (ds) and single-stranded (ss) DNA junctions flanking RNA:DNA hybrids may help RNase H find the hybrid substrates in long genomic DNA. Our study provides new insights into the multifunctionality of RNase H, elucidating unprecedented roles of junctions and ssDNA overhang on RNA:DNA hybrids.RNase H is involved in fundamental cellular processes and is responsible for removing the short stretch of RNA from Okazaki fragments and the long stretch of RNA from R-loops. Defects in RNase H lead to embryo lethality in mice and Aicardi-Goutieres syndrome in humans, suggesting the importance of RNase H. To date, RNase H is known to be a non-sequence-specific endonuclease, but it is not known whether it performs other functions on the structural variants of RNA:DNA hybrids. Here, we used   RNase H participates in many fundamental genetic processes, such as replication and transcription ,2. DurinOver the past decades, many crystal structures of RNase H have been determined, from sources ranging across T4 bacteriophages  to bacteBacillus halodurans RNase HI were solved in complex with an RNA:DNA hybrid (Escherichia coli) (E. coli). RNase HI in E. coli is a homolog of RNase H1 in the mitochondria and the nucleus of eukaryotes. In particular, E. coli RNase HI is structurally very similar to the C-terminal catalytic domain (RNase HC) of human RNase H1 except for the N-terminal hybrid-binding domain (HBD)   whereas in (HBD) . From heNucleic acid enzymes typically recognize the structural features of their substrates, utilize the chemical properties of nucleic acids, and perform organized reactions, i.e. a series of cleavage reactions using directional translocation along polarized nucleic acids . For this reason, interaction and catalysis by RNase H should also be influenced by the basic structural features of substrates, such as the 3′ and 5′ ssDNA overhangs of R-loops, different duplex junctions, and the chimeric junction of an RNA–DNA strand  in the genomic DNA. To date, however, structural and biochemical studies on RNase H have focused mainly on simple RNA:DNA hybrids, not on hybrids containing an ssDNA overhang, different duplex junctions or chimeric junctions of the RNA–DNA strand .Many intriguing questions arise as to how RNase H recognizes and responds to structural distortion since chimeric RNA–DNA:DNA hybrids become gradually distorted from the A form of RNA:DNA to the B form of DNA:DNA. Like some enzymes that utilize the hydrolysis of phosphate groups to move in one direction along polarized nucleic acid strands ,25, doesRecent progress in single-molecule (sm) methods has allowed us to explore physiologically relevant dynamic activities such as heterogeneity of activity , the anaE. coli RNase H gene was inserted into the pET28a vector, which contains a 6-histidine tag. The cloned vector was confirmed by DNA sequencing, and RNase H was expressed in E. coli cells. The fusion plasmid construct was transformed into BL21-Star E. coli and expressed in 1 L of LB. Bacterial cultures were grown to an OD600 of 0.5. Then, IPTG was added at a final concentration of 1 mM. After shaking for 14–16 hours at 18°C, bacteria were harvested in a rotor at 5000 × g, resuspended in 40 ml of buffer (20 mM Tris–HCl (pH 7.5), 500 mM NaCl) and lysed by sonication. The cell lysate was centrifuged for 30 min at 35 000 × g. After the supernatant was filtered, the sample was loaded into a HisTrap FF column (5 ml) . The protein was eluted through a gradual increase in imidazole , and purified RNase H was stored in buffer containing 50 mM KCl, 10 mM Tris–HCl (pH 7.4), 0.1 mM EDTA, 1 mM DTT, 200 μg/ml BSA and 50% glycerol.The E. coli RNase H has been successfully used in protein folding and functional studies without affecting its activity . Two amine-modified DNA strands were labeled with Cy3 and Cy5, and the nonhydrolyzed strand was constructed by ligating the two oligos using T4 ligase at room temperature for 3 h. The ligated strand was purified from unreacted oligonucleotides via 15% PAGE. The fluorescently labeled nonhydrolyzed DNA was annealed with the 5′ hydrolyzed strand in 10 mM Tris–HCl (pH 8.0) with 50 mM NaCl by heating for 2 min at 85°C and cooling slowly to room temperature. Sequence and modification information is shown in Single-molecule experiments were performed with a prism-type TIRF  microscope. Briefly, the fluorescence emission light from the donor (Cy3) and acceptor (Cy5) fluorophores was transmitted by a water-immersion objective lens  and then collected through a 550-nm longpass filter to filter out scattered light from a 532 laser. The fluorescence emission spectrum was further divided into donor (green) and acceptor (red) signals with a dichroic mirror with a 630-nm cutoff (Chroma) and recorded by a back-illuminated electron-multiplying charge-coupled device  with the time resolution of 100-ms (for degradation assays) and 500-ms (for binding assays).2, 100 μg/ml BSA, 1 mg/ml Trolox, 1 mg/ml glucose oxidase, and 0.04% mg/ml catalase. We made the chamber with a coverslip and slide using epoxy and double-sided tape. Holes in the slide were made for the entrance and exit, and a syringe was connected with the holes using tubes. Because the pipette tip containing reaction buffer was fixed to the entrance hole, when the syringe was pulled, the solution in the tip was introduced into the chamber. When RNase H  was added to the reaction buffer, the degradation reaction began.The substrate was immobilized on the quartz surface covered with PEG through interaction between biotin labels on DNA and Neutravidin (Pierce) on the PEG surface. For single-molecule imaging, we injected ∼10 pM hybrid substrates into the chamber. The reaction buffer contained 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 10 mM MgCl2, 100 μg/ml BSA, 1 mg/ml Trolox, 1 mg/ml glucose oxidase, 0.04% mg/ml catalase, and 5 nM RNase H for binding only. For the removal of free RNase H and a single reaction, we injected washing buffer containing 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 10 mM MgCl2, 100 μg/ml BSA, 1 mg/ml Trolox, 1 mg/ml glucose oxidase and 0.04% mg/ml catalase.The substrate was immobilized in the same way described in the method for the single-molecule assay of degradation by the multiturnover reaction. After immobilization, we injected binding buffer containing 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 1 mM CaCl2 as a binding cofactor to prevent the degradation reaction. Binding buffer containing 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 10 mM CaCl2, 100 μg/ml BSA, 1 mg/ml Trolox, 1 mg/ml glucose oxidase, 0.04% mg/ml catalase and 0.2 nM C-terminus Cy5-labeled RNase H was injected into the chamber. We observed binding events through FRET whenever Cy5-labeled RNase H bound to the Cy3-labeled substrate.The substrate was immobilized as described in the single-molecule degradation assay by the multiturnover reaction. To observe binding, we used Cy3-labeled substrate and Cy5-labeled RNase H instead of Cy3- and Cy5-labeled substrate and nonlabeled RNase H. We used CaCl2, 75 mM KCl, 50 mM Tris–HCl (pH 8.3) and 100 μg/ml BSA. The sample was incubated for 20 s . Substrates for the gel assay were prepared as described in Cy3 labeling and annealing. For a degradation reaction, 100 nM substrate was mixed with 5 nM RNase H in 10 μl of buffer containing 10 mM MgClfor 20 s  and 5 mifor 20 s  at room n . The errors of degradation time, count, frequency, running time, and fraction indicate the standard error of the mean (s.e.m.): s.e.m. = σ/√ Figures . The per Figures . The staE. coli RNase H was site-specifically labeled with a Cy5 acceptor (termed Cy5-RNase H) at the C-terminus via cysteine engineering , which recapitulated the two-dimensional histogram for the pair of FRET values before and after each binding event  were attained from [average time of unbound]/ [(average time of bound) × (protein concentration)] ] ,49. Theiration)] : 3′ ssDNration)] . The affration)] ,22.2+ were plotted to TDP for the hybrids carrying the 3′ ssDNA overhang. For the 3′ overhang-bearing RDH (i.e. 3′ORDH), the primary binding site appeared at the ss-ds junctions (termed 3′OJ) consisting of a 3′ ssDNA overhang and RNA:DNA heteroduplex, represented by the high FRET, E = 0.77, and the secondary binding site appeared at the CJ, represented by the low FRET, E = 0.48  and acceptor (Cy5) were conjugated at the DNA sites opposite the 5′ end of ssRNA and the CJ of the RNA–DNA strand, respectively , which allowed us to measure the degree of degradation per attempt. The RNA hybrid was incubated with RNase H (5 nM) in the presence of CaH Figure  so that H Figure , the SR H Figure , indicatH Figure .2+, the prebound Cy5-RNase H processively degraded 3′ ORDH while moving away from its Cy3 position, progressively decreasing FRET without any signal loss  of the 3′OJ and 5′OJ were ∼0.87 and 2.34 nM, respectively, consistently showing a difference of ∼3-fold  to high FRET (E = 0.7) with increasing enzyme concentration  in RNA:DNA hybrids function as structural recognition sites for RNase H Figures  and 3. TKd) than to the inner region of RNA:DNA hybrids . Our study offers novel mechanistic insights into the recognition of RNase H in RNA:DNA intermediate structures containing dsDNA flanking junctions. We also found that the 3′ ssDNA overhang, if present, facilitates the overall reaction, making RNase H a processive exoribonuclease. Furthermore, RNase H successively degrades RNA while translocating along the RNA:DNA hybrid without falling off. This asymmetric bifunctional enzymatic activity makes RNase H unique in efficiently removing RNA moieties during dynamic replication, transcription, and repair processes. During nucleic acid metabolism, 3′ overhang and 5′ overhang strands are frequently produced by various nucleases, suggesting that this type of interaction may occur with other DNA- and RNA-interacting proteins, allowing these nucleases to perform a variety of functions.The data that support the findings of this study are available from the corresponding author upon reasonable request.gkab1064_Supplemental_FileClick here for additional data file.


Opisthorchis felineus worms. Promising agents have been found among both natural and synthetic compounds. The mitochondria-targeted antioxidant SkQ1 [10-(6′-plastoquinonyl)decyltriphenylphosphonium] in motility assays was as effective  as praziquantel (IC50 0.47–1.4 μM), and SkQ1 was significantly more effective than praziquantel in mortality assays. Moreover, extensive tegument damage of the adult fluke was revealed after SkQ1 treatment. Flavonoids manifested potency too, with IC50 values in a micromolar range (5.1–17.4 μM). Other natural and synthetic compounds tested against helminths were significantly less effective than praziquantel. Results of our study indicate that SkQ1 and flavonoids have high anthelmintic activities against the liver flukes. We propose that structure–activity relationship research might be worthwhile based on the structures of the most effective substances.Currently, molecular parasitologists are searching for new agents against trematodiases. Redox metabolism is important for parasites as far as long-lived adult parasites inside a mammalian host are exposed to redox challenges. Antioxidants have been poorly studied as anthelmintic agents, in particular against the foodborne trematodes. Study of in vitro anthelmintic activity of nonenzymatic natural and synthetic antioxidants of various chemical structures was performed using standard motility and mortality assays against juvenile and adult  Clonorchis sinensis (C. sinensis), Opisthorchis viverrini (O. viverrini) and Opisthorchis felineus (O. felineus) affect millions of people and have a major socioeconomic impact worldwide. These trematodiases have similar pathogenesis. Humans become infected by eating undercooked or raw Cyprinidae fish -sulfide), CO4 propyl]-disulfide), and betamide (N-[3-oxo-20(29)lupen-28-oil]-3-aminopropionic acid) were synthesized and provided by Vorozhtsov Institute of Organic Chemistry SB RAS . The chemicals were dissolved in dimethyl sulfoxide (DMSO)  to obtain 10 mM stock solutions. 2 incubator [Newly excysted metacercariae (NEM) were hatched from metacercariae ,9,28. Flncubator ,9,10.50), we tested the following concentrations of compounds: 0.001, 0.01, 0.1, 1, 10, 100, and 500 µM, the dilution factor was 1:200. The solvent concentration across different compound concentrations was 0.5%. Four to five adult worms and 30–40 NEM were used per well. The experiments were repeated three times with two or three biological replicates for each concentration. As control groups, we incubated flukes in the medium with 0.5% DMSO. After 24 h, the motility was evaluated under the light microscope  on a motility scale from 0 to 3  as described previously [50 and standard error (SE) values (‘drc 3.0-1’ R package). The ANOVA lack-of-fit test was performed to assess the hypothesis that a proposed regression model fits the data well. Worm viability was evaluated under an inverted microscope (Axiovert 40CFL) equipped with a camera  after 24 h of treatment (magnification 10–50×). The worms were classified as dead if they had a dark color and no movement was observed for 2 min ,28. For eviously ,28. DataFor microscopic examination of tegument, adult flukes after the treatment with DMSO, 1 µM PZQ, 10 µM SkQ1 were fixed, processed and stained with hematoxylin and eosin dye as previously described .To estimate the mortality rates, NEM were treated with one of the following agents: 1 µM PZQ, 10 µM SkQ1, 1 µM SkQ1, 10 µM flavone, 10 µM quercetin, 10 µM silymarin, 10 µM resveratrol, or 0.5% DMSO and were incubated at 37 °C for 15 days. The effect of each drug was evaluated three times. To estimate the mortality rates, Kaplan–Meier survival curves were built using the ‘survival’ (v.2.38) R package. Finally, statistical significance of a difference in the survival log-rank (Mantel–Haenszel) test within each pair of samples was calculated. During the past decades, a lot of research has been carried out around antioxidants and their influence on health. It has been emphasized that the balance between oxidation and antioxidation is critical for a healthy biological system. Low doses of antioxidants may be beneficial to this system, but high quantities may disrupt the balance . The limTo conclude, SkQ1 and other antioxidant substances appear to have an anthelmintic activity against liver flukes in motility and mortality assays. We propose that structure–activity relationship research might be worthwhile based on the structures of the most effective substances. Studies should also be undertaken to assess possible synergy of combinations of these substances with PZQ against parasites, other trematode species, and liver fluke infections in laboratory animals.