To date, all clinical and neuropathologically confirmed vCJD cases have been Met129 homozygous, with the exception of 1 recently reported Met/Val heterozygous case. Here, we found that transgenic mice homozygous for Val129 Hu-PrP show severely restricted propagation of the BSE prion strain, but this constraint can be partially overcome by adaptation of the BSE agent to the Met129 Hu-PrP. In addition, the transmission of vCJD to transgenic mice homozygous for Val129 Hu-PrP resulted in a prion with distinct strain features. These observations may indicate increased risk for vCJD secondary transmission in Val129 Hu-PrP–positive humans with the emergence of new strain features.Bovine spongiform encephalopathy (BSE) is the only known zoonotic prion that causes variant Creutzfeldt-Jakob disease (vCJD) in humans. The major risk determinant for this disease is the polymorphic codon 129 of the human prion protein (Hu-PrP), where either methionine (Met Sc) of prion protein (PrP), which is converted from the normal cellular isoform (PrPC)  is considered by strong epidemiologic, pathologic, and molecular evidence to be a likely consequence of human dietary exposure to the bovine spongiform encephalopathy (BSE) agent , where methionine (Met) or valine  can be encoded, strongly affects susceptibility to human prion diseases , and all efforts were made to minimize suffering. Experiments were approved by the Committee on the Ethics of Animal Experiments of the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria .–We used 11 isolates from different sources in this study (129 (TgMet129) mouse line expressing human Met129-PrPC variant  mouse line expressing human Val129-PrPC variant  transgenic mouse line obtained by mating TgMet129 and TgVal129 mice (C (around 4-fold the level of expression in the human brain) on a mouse PrP null background. We performed additional inoculations in HuPrP-Tg362-Val129, a transgenic mouse line expressing 8-fold the level of PrPC expression in human brain   HuPrP-Tg340-Metres) by Western blot. We anesthetized individually identified mice, 6–7 weeks of age, with isoflurane and inoculated them with a 2-mg equivalent of brain homogenate in the right parietal lobe by using a 25-gauge disposable hypodermic needle. We observed mice daily and assessed neurologic status 2 times per week. When progression of a TSE disease was evident or at the established experimental endpoint (700 days postinoculation [dpi]), we euthanized the animal for ethical reasons and performed necropsy, excising the brain. We then fixed part of the brain by immersion in neutral-buffered 10% formalin  and used the tissue for quantifying spongiform degeneration by histopathology. We froze the remaining tissue at −20°C and used it to determine the presence of disease-associated proteinase K (PK)–resistant PrP . Based on a previously described protocol . Immunoblots were developed with enhanced chemiluminiscence ECL Select . Images were captured using the ChemiDoc WRS+ System (Bio-Rad) and processed using Image Lab 5.2.1 software (Bio-Rad).We homogenized frozen brain tissues (175 ± 20 mg) in 5% glucose in distilled water in grinding tubes  adjusted to 10% (wt/vol) by using a TeSeE Precess 48TM homogenizer (Bio-Rad), according to the manufacturer’s instructions. We determined presence of PrPWe performed procedures for the histopathological analysis of mouse brains as previously described  or Val  at codon 129 of human PrP or were their F1 cross . These mouse models expressed similar human PrP levels, ≈4-fold more than that seen in uninfected human brain tissue (res accumulation in control mice inoculated with TSE-free control brain homogenate. The 3 human transgenic mouse models were readily infected when inoculated with sporadic CJD (sCJD) (129 type 1 (Hu-sCJD MM1) and Val129 type 2 (Hu-sCJD VV2)  . The 2 s129 mice  mouse line (129 (8×) mice showed any evidence of infection after challenge with the different BSE isolates . On first passage, 100% of the TgMet129 mice developed clinical disease in response to all inocula in the panel  blots in the mouse brains  analysis (129 mice inoculated with Hu-vCJD2 (that remained apparently uninfected) to BoPrP-Tg110 mice showed evidence of subclinical infection. These subpassages led to a mean incubation time of 371 ± 5 dpi and to propagation of PrPres that was detectable by WB in 100% of animals , characterized by low size fragments (19-kDa fragment for the aglycosyl band) and prominent diglycosylated species on WB  and our vCJD-TgVal129 PrPSc, we performed a biochemical characterization by WB and found no molecular profile differences in PrPres from the various mouse lines   , lane 8.PRNP codon 129 genotypes. Because a high expression level of PrP in transgenic mice directly influences prion disease susceptibility and incubation time, these transgenic mice have an advantage over knock-in mice for evaluating these features in the different human PrP genotypes. In addition, the 3 mouse models used in our study have equivalent PrP expression levels, making them suitable for studying comparative susceptibilities across the different PRNP codon 129 genotypes.We report a detailed comparison of the transmission properties of BSE and vCJD prions among humanized transgenic mice with different 129 homozygous individuals might be susceptible to a sheep or goat BSE agent to a higher degree than to cattle BSE and that these agents might transmit with molecular and neuropathological properties indistinguishable from those of vCJD  was clinically transmitted to 1 of 10 TgMet/Val129 mice directly, adaptation of the BSE agent to human PrP Met129 sequence and subsequent inoculation of the resultant vCJD prions to TgMet/Val129 mice produced a 100% attack rate. However, we did not detect clinical prion disease, supporting a slower rate of vCJD conversion compared with that among TgMet129 mice. This slow but potential conversion rate in TgMet/Val129 mice correlates well with the single vCJD case of a human carrying the PrP Met/Val129 genotype  might be caused by differences in prion titer between inocula. This assessment was strengthened after the transmission of both vCJDs to TgMet129 mice, in which a shorter incubation period was observed in animals inoculated with Hu-vCJD2. A certain variability in subclinical transmissibility and incubation time between different vCJD isolates is not uncommon, as has been previously reported  prions, and the propagated agents might transmit with molecular and neuropathological properties distinguishable from those of type 4 PrPres. Although the resultant type 5 PrPSc shares the same fragment sizes as those of type 2 PrPSc, the 2 PrPSc types can be distinguished by the predominance of the diglycosylated glycoform associated with type 5 PrPSc. Overall, our results indicate that human Val129-PrP polymorphic variant is a strong molecular protector against BSE zoonotic transmission but fails to prevent human-to-human vCJD transmission. Because potential late-onset vCJD cases could appear in the population  mice.Transmission of Ca-BSE bovine spongiform encephalopathy–derived isolates adapted in different human prion protein polymorphic variants to BoPrP-Tg110 mice and


The transmission of classical bovine spongiform encephalopathy (C-BSE) through contaminated meat product consumption is responsible for variant Creutzfeldt-Jakob disease (vCJD) in humans. More recent and atypical forms of BSE (L-BSE and H-BSE) have been identified in cattle since the C-BSE epidemic. Their low incidence and advanced age of onset are compatible with a sporadic origin, as are most cases of Creutzfeldt-Jakob disease (CJD) in humans. Transmissions studies in primates and transgenic mice expressing a human prion protein (PrP) indicated that atypical forms of BSE may be associated with a higher zoonotic potential than classical BSE, and require particular attention for public health. Recently, methods designed to amplify misfolded forms of PrP have emerged as promising tools to detect prion strains and to study their diversity. Here, we validated real-time quaking-induced conversion assay for the discrimination of atypical and classical BSE strains using a large series of bovine samples encompassing all the atypical BSE cases detected by the French Centre of Reference during 10 years of exhaustive active surveillance. We obtained a 100% sensitivity and specificity for atypical BSE detection. In addition, the assay was able to discriminate atypical and classical BSE in non-human primates, and also sporadic CJD and vCJD in humans. The RT-QuIC assay appears as a practical means for a reliable detection of atypical BSE strains in a homologous or heterologous PrP context. Sc, an abnormal isoform of the host-encoded cellular prion protein (PrPc). The infectious agent is mainly, if not solely, composed of abnormal PrPSc and is capable of converting cellular PrPc into PrPSc in an autocatalytical manner [Sc, molecular features of the protease resistant fragment (PrPres) can be evidenced by Western blot.Prion diseases are fatal transmissible disorders affecting humans and animals. These neurodegenerative diseases are characterized by brain vacuolization, neuronal loss and accumulation of PrPl manner . After pres observed in Western blot [res molecular changes could be observed in some conditions [Among animals, the classical bovine spongiform encephalopathy (C-BSE) affects cattle. The C-BSE epidemic in the 1980s became a major matter of concern for human health when the variant of Creutzfeldt-Jakob disease (vCJD) appeared as the result of a C-BSE foodborne transmission to humans –4. Sinceern blot . The annern blot . When trern blot –9 and trern blot , 11, L-Bern blot , 12. Wheern blot  and tranern blot , 15, spenditions . Taken tAmong recent methods developed to amplify prions in vitro, the real-time quaking-induced conversion (RT-QuIC) assay allows the sensitive detection of prion seeding activity in numerous tissues of animal and human origins –26. The In two recent studies, this test was used successfully for the discrimination of a few C-BSE and L-BSE samples of cattle , and forA written informed consent for autopsy and research use was provided by patient’s relatives, according to the French regulation . The brain tissues with the corresponding written informed consent are referred for postmortem diagnosis and research to the French National Neuropathological Network for CJD (funded by the French Government) and to the French National Centre of Reference for prions . No approval by local ethics committee is required during this procedure.Agence Nationale de Sécurité Sanitaire de l’Alimentation, de l’Environnement et du Travail . In this study, 13 H-BSE and 14 L- BSE isolates, encompassing all the atypical BSE cases collected during the period 2000–2010, were analyzed by RT-QuIC. Fifteen C-BSE cases collected during this period were also included. Among samples collected from fallen stock or after several freeze-and-thaw cycles, autolysis was often observed, affecting 10 out of 13 H-BSE samples, 11 out of 14 L-BSE samples and 6 out of 15 C-BSE samples , their PrPres type (type 1 or 2), and according to the migration pattern of PrPres on Western blot. Patients were referred to the French National Reference Center for Unconventional Transmissible Agents for CJD, and the diagnosis was confirmed biochemically and neuropathologically. Brain samples from cynomolgus macaques previously inoculated via intracerebral and oral routes with classical and atypical L-BSE isolates [Samples from primates and cattle existed before the study began. BSE brainstem samples were collected during the active surveillance in France and confirmed by discriminatory western blot by the  samples . Brainstisolates , 30 wereHuman recombinant full length PrP (codon 129M)  and bovine recombinant full length PrP  were purified according to the protocol published previously [® 24 instrument  for 45 s at speed 6.5. Gross cellular debris were removed from brain homogenates (BH) after a centrifugation at 2000 g for 2 min. Supernatants were collected and stored at -80°C until use.The tissues were prepared in PBS (Sigma-Aldrich) containing 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, and Complete Protease Inhibitor Cocktail (Roche) to give a final tissue concentration of 10% (w/v). The homogenization was performed in 2ml centrifuge tubes containing ceramic beads using a FastPrepSc, brain homogenates (BH) from CJD patients were normalized using known concentrations of recHuPrP. The samples containing PrPres were digested with PK 100μg/ml for 1h at 37°C and loaded onto Novex 4–12% Bis-Tris acrylamide gels (Life Technologies), alongside dilutions of recHuPrP (ranging from 10 to 1.25 ng). To compare the detection of the studied prion strains in the different groups of affected individuals , brain homogenates were analyzed by Western blot (WB) as previously described [res in samples was adjusted with a further dilution to match the amount detected in the sample with the lowest PrPres signal at the determined dilution.To assess the analytical sensitivity of our RT-QuIC assay for the detection of sCJD and determine the minimal amount of detectable PrPescribed . The amores. To obtain an estimated amount of PrPres in 2 μl equivalent to 100 fg of recHuPrP, the corresponding 100% brain tissue dilutions varied from 10−6  to 5x10-7 . For BSE isolates, all the samples were tested by RT-QuIC at the same dilution (10−4), regardless of the PrPres level in the sample. A Western blot analysis with TeSeE confirmatory Western blot kit (Bio-Rad) was done to compare relative quantity of PrPres between groups  and to confirm the presence of PrPres when no seeding activity was detected by RT-QuIC.After electrophoresis, the separated proteins were transferred to nitrocellulose membrane and immunoblotted with anti-PrP mAb 3F4 (Eurogentec) for human samples. It was followed by an incubation with a secondary antibody coupled with horseradish peroxidase (HRP). The HRP activity was revealed using ECL (GE Biosciences), and the blots were exposed to ECL Hyperfilms (GE Biosciences). The films were scanned using a Bio-Rad GS800 densitometer and analyzed using Bio-Rad Quantity One software. The densities of the single Western blot band corresponding to recHuPrP were compared with the combined densities of the 3 bands corresponding to PrPThe RT-QuIC assay was prepared as described previously , 33. SamAll RT-QuIC experiments were performed at least three times and produced comparable results. At pertinent time points, the statistical significance of the difference between mean fluorescence or lag phases of analyzed groups was assessed using the non-parametric, unpaired t- test with unequal variance (Welch correction), using GraphPad Prism software v6.0 .res Western blot results for each BSE group are illustrated in res level was observed in C-BSE samples. A typical run was performed using 2 plates, in which were included the 8 negative samples and a half of each BSE group. In our conditions, negative and classical BSE samples could not be differentiated using recHuPrP  to ascertain that the lack of C-BSE amplification we observed was not due to the use of recHuPrP. Similar results were obtained with recBovPrP. While C-BSE and negative samples remained undistinguishable  used to seed the reaction, vCJD brain homogenates proved less efficient to initiate RT-QuIC reactions, and only a sensitivity down to a 10−7 brain dilution  could usually be achieved in our conditions  .res content. Thus, our study confirms previous results obtained with 5 L-BSE and 4 C-BSE natural cases by Orru et al [In this study, we assessed the power of discrimination of BSE strains from cattle using RT-QuIC. Indeed, in two recent studies, this assay was used successfully for the discrimination of a few C-BSE and L-BSE samples from cattle , and forru et al  and withru et al , using lsc glycotypes, neuropathology and PrPsc deposition different from those observed in classical BSE [Atypical cases of BSE putatively represent sporadic forms of prion disease in cattle, with PrPical BSE , 12. In ical BSE . Using sical BSE  or humanical BSE , withoutical BSE . Likewisical BSE . In our res formation [in vitro formation of PrPres [sc assemblies and therefore on the related seeding activity. For example, the H187R mutation in the human PrP has a dramatic effect on the protein folding, resulting in a markedly increased propensity to oligomerize [RT-QuIC discrimination may rely on differences in abnormal PrP assemblies sustaining distinct seeding activities that may vary with several factors . We were able to discriminate brain samples from macaques inoculated with C-BSE and with L-BSE. Surprisingly, in macaques, C-BSE showed the most efficient seeding activity while it was relatively inefficient in humans. Differences in PrP amino acid sequence might contribute to this discrepancy. Mature forms of bovine and human PrP share 91.3% of amino acid sequence, and bovine and cynomolgus macaques PrP only 88.7%. Different regions of PrP have been proposed as key domains for fibrillization, such as the S1H1S2 region , 35 or tormation  and becaf PrPres . Other vgomerize .However, the unpredictable manner of the biological changes of prion strain properties during interspecies transmission has been extensively described –44. In mSc fibers which could modulate the seeding activity of these tissues. However, plaques are also observed in the brain of vCJD patients, and vCJD brain homogenates show a poor seeding activity. Another characteristic of C-BSE in macaques is the high proportion of diglycosylated PrPres, but C-BSE or vCJD PrPres share the same feature, and yet are not associated with an efficient seeding activity. Likewise, the Western blot type of PrPres as defined by the molecular mass of the unglycosylated PrPres after proteinase K digestion cannot account for these different seeding activities, since PrPres type 2 is distributed in both groups with efficient  and inefficient (vCJD) seeding properties. It was intriguing that PrPsc from all the studied natural diseases with a sporadic or presumably sporadic origin  showed a high level of seeding activity, unlike peripherally acquired diseases due to the C-BSE agent . To investigate the role of the peripheral route on the selection of PrPSc species with seeding activity, we analyzed brain homogenates from iatrogenic CJD cases secondary to growth hormone treatment, which are acquired prion diseases and result from a human-to-human CJD transmission. We found similar and efficient seeding activities for sCJD MM1 and iCJD-hGH cases using RT-QuIC. Moreover, we also found similar and efficient seeding activities in brain homogenates from non-human primates inoculated intracerebrally and via the oral route with C-BSE or L-BSE. Altogether, our data do not support the peripheral route as a main factor influencing the selection of PrPsc species with low seeding activity. In our hands, it appears that this poorly efficient RT-QuIC profile was limited to the C-BSE agent, which propagated in cattle and humans.A main feature of C-BSE-infected brain tissue from various animal models including macaques is the presence of amyloid plaques –52 compoSc from a large series of 27 atypical BSE isolates within hours, and provides a promising tool for the diagnosis of these natural diseases and for their discrimination from C-BSE in a homologous and heterologous PrP context.To conclude, we showed that RT-QuIC detects PrPS1 Fig−4 dilutions of bovine tissue (brainstem), using human recombinant protein. (A) and (D), classical BSE and uninfected bovine tissues. (B) and (E), classical BSE and atypical H-BSE. (C) and (F), classical BSE and atypical L-BSE. Each point represents the mean value of 3 replicate relative fluorescence unit readings.RT-QuIC reactions were seeded with 10(PDF)Click here for additional data file.S2 FigA), classical BSE isolates and uninfected bovine samples. (B), classical BSE and atypical H-BSE isolates. (C), classical BSE and atypical L-BSE isolates. Each point represents the mean value of 3 replicate relative fluorescence unit readings, which were averaged over the number of animals in each group. Error bars represent the mean standard deviation (SD). Vertical dashed lines indicate a statistically significant difference of signal between the test groups. *, p<0,05; ****, p<0,0001.Average data and statistical significance of the individual results presented in (PDF)Click here for additional data file.S3 Fig−4 dilutions of bovine tissue (brainstem), using bovine recombinant protein. (A) and (D), classical BSE isolates and uninfected bovine samples. (B) and (E), classical BSE and atypical H-BSE isolates. (C) and (F), classical BSE and atypical L-BSE isolates. Each point represents the mean value of 3 replicate relative fluorescence unit readings.RT-QuIC reactions were seeded with 10(PDF)Click here for additional data file.S4 FigHomogenates were subjected to proteinase K digestion and serial dilutions were detected by immunoblotting using Sha31 (primates) or 3F4 (CJD patients) monoclonal antibodies.(PDF)Click here for additional data file.


Pre- and post-natal factors can affect brain development and function, impacting health outcomes with particular relevance to neurodevelopmental diseases, such as autism spectrum disorders (ASDs). Maternal obesity and its associated complications have been related to the increased risk of ASDs in offspring. Indeed, animals exposed to maternal obesity or high fat diets are prone to social communication impairment and repetitive behavior, the hallmarks of autism. During development, fatty acids and sugars, as well as satiety hormones, like insulin and leptin, and inflammatory factors related to obesity-induced low grade inflammation, could play a role in the impairment of neuroendocrine system and brain neuronal circuits regulating behavior in offspring. On the other side, post-natal factors, such as mode of delivery, stress, diet, or antibiotic treatment are associated to a modification of gut microbiota composition, perturbing microbiota-gut-brain axis. Indeed, the interplay between the gastrointestinal tract and the central nervous system not only occurs through neural, hormonal, and immune pathways, but also through microbe-derived metabolic products. The modification of unhealthy perinatal and postnatal environment, manipulation of gut microbiota, nutritional, and dietary interventions could represent possible strategies in preventing or limiting ASDs, through targeting inflammatory process and gut microbiota. Over the past decades, the frequency of autism diagnoses has been steadily climbing and has increased the interest of the scientific community  ailments and changes in microbiota composition  and hormones (leptin and insulin) occurring in obese mothers are possible events involved in the impaired development of offspring , the autonomic nervous system (both sympathetic and parasympathetic branches), the neuroendocrine and neuroimmune systems, the enteric nervous system (ENS), and the gut microbiota. All these components intercommunicate via the immune system, the vagus nerve, and other host microbe interactions, and influence each other, constituting a complex network. The main neuroendocrine pathway is the hypothalamic-pituitary-adrenal (HPA) axis, activated in response to various physical and psychological stressors. A crucial player in this communication between peripheral signals and the CNS is the gut microbiota (GM), therefore, this interplay was re-named microbiota-gut-brain axis, viewed as a bidirectional communication system between gut microbes and CNS , a mild stress lead to a higher release of corticosterone and adrenocorticotrophic hormone (ACTH) compared to mice with common and no pathogen bacteria (specific pathogen free mice). Moreover, the administration of GM capability to influence the brain activity is based on the production of neuroendocrine hormones and neuroactive compounds that play a pivotal role in shaping cognitive networks underlying social cognition, emotion, and behavior  exposure, gut inflammation, altered microbiota, and ASD-like behavioral abnormalities in male offspring were reported  proved to be a key treatment to ameliorate alterations in commensal microbiota, to restore intestinal permeability and cytokine production and to improve behavioral abnormalities , free amino acids (FAAs), and phenol compounds , in neuropsychological disorders has been deeply clarified  is associated with less incidence of numerous GI disorders. On the other hand, several studies show that nutritional deficiencies of autistic patients are filled with the supplementation of vitamins and minerals, fatty acids ω-3, probiotics, in combination with pharmacological and psychological interventions, even if supplementation interventions show contrasting, but promising results.A typical strategy to decrease food related effects in ASD is a gluten-free and casein-free (GFCF) diet.GFCF diet consists in the elimination of food containing gluten, and products containing gluten trace amounts. This diet also eliminates casein, a protein present in cow milk and dairy products. The absence in this diet of milk and dairy products leads, however, to calcium, phosphate, and vitamin D deficiency. Therefore, nutrition specialists usually recommend soy or rice milk as substitutes for cheese  possibly due to peptidase deficiencies. This alteration may result in excessive opioid activity in the CNS, altering its function. In particular, gluten- or casein-derived peptides are suspected to be involved in ASD, resembling opioid-like molecules , introduced by Gotschall , represeThis diet largely recommends monosaccharides, whose origin is fruit, vegetables, and honey, whereas it removes complex carbohydrates. The formulation of this diet is based on the evidence that autistic patients have an abnormality in carbohydrate digestion and adsorption, causing residual food accumulation that represents a breeding ground for pathogenic intestinal flora , eicosapentaenoic acid , and docosahexaenoic acid , are crucial for brain development and cognitive and memory functions Das, .Maternal intake of PUFAs improved memory in the progeny, suggesting that, supplementation of PUFAs needs to start during pregnancy and continue after delivery until brain development is complete in adolescence. In agreement, breastfeeding improves brain growth and development and memory, since breast milk is richer in AA and DHA compared with formula . On the basis of microbiota involvement in ASD etiology, probiotics have been considered as a therapeutic tool able to impact brain development, and behavior. Therefore, the rationale of the use of probiotics, has been to re-establish the healthy equilibrium of GM altered in ASDs. One of the first evidence showing the impact of GM on ASDs has been demonstrated by Sandler et al.  plantarum WCFS1 for the following 3 weeks, and the other receiving first the probiotic and then the placebo  for 3 weeks and L. reuteri or L. rhamnosus in order to prevent GI colonization by Candida species, the authors reported the reduction of Candida in stool from treated infants with both probiotics, as well as GI symptoms by L. rhamnosus administration; notably, all treated newborns showed lower incidence of poor neurological outcomes compared to untreated control group, evaluated up to 12 months by the Hammersmith Infant Neurological examination  is a procedure through which patients receive the fecal microbiota from healthy donors. Generally, this practice is beneficial in ailments where dysbiosis plays a pivotal role, since it aims to restore GM homeostasis. Obviously, GI disorders, such as inflammatory bowel diseases and irritable bowel syndrome, are the most common ailments suggested for this intervention, even if also other disorders, like autoimmune disease and obesity has been tested with beneficial effects , in order to correct dysbiosis and restore a healthy conversation between gut and brain. This would partially lessen both gastrointestinal and neurobehavioral symptoms.CC, AL, FL, MPM, AC, and GM wrote the paper. CC, MPM, and GM supervised the review editing. GM decided the overall structure of the review, coordinating the working group.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Anacyclus pyrethrum (L.) is a plant widely used in Moroccan traditional medicine to treat inflammatory and painful diseases. The objective of the present study was to evaluate the antinociceptive, anti-inflammatory and antioxidant activities of aqueous and methanol extracts of Anacyclus pyrethrum roots (AEAPR and MEAPR). The anti-inflammatory effect of AEAPR and MEAPR was determined in xylene–induced ear edema and Complete Freund’s Adjuvant (CFA)-induced paw edema. The antinociceptive activity of AEAPR and MEAPR  administered by gavage was examined in mice by using acetic acid-induced writhing, hot plate, and formalin tests, and the mechanical allodynia were assessed in CFA-induced paw edema. In addition, the in vitro antioxidant activities of the extracts were determined by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method, ferric reducing power and β-carotene-linoleic acid assay systems. AEAPR and MEAPR produced significant reductions in CFA-induced paw edema and xylene-induced ear edema. A single oral administration of these extracts at 250 and 500 mg/kg significantly reduced mechanical hypersensitivity induced by CFA, which had begun 1 h 30 after the treatment, and was maintained till 7 h. Chronic treatment with both extracts significantly reduced mechanical hypersensitivity in persistent pain conditions induced by CFA. Acute pretreatment with AEAPR or MEAPR at high dose caused a significant decrease in the number of abdominal writhes induced by acetic acid injection , a marked increase of the paw withdrawal latency in the hot plate test, and also a significant inhibition of both phases of the formalin test. This antinociceptive effect was partially reversed by naloxone pretreatment in the hot plate and formalin tests. Additionally, a significant scavenging activity in DPPH, reducing power and protection capacity of β-carotene was observed in testing antioxidant assays. The present study suggests that AEAPR and MEAPR possess potent anti-inflammatory, antinociceptive and antioxidant effects which could be related to the presence of alkaloids and phenols in the plant. In addition, the antinociceptive effect of APR extracts seems to partly involve the opioid system. Taken together, these results suggest that Anacylcus pyrethrum may indeed be useful in the treatment of pain and inflammatory disorders in humans. Chronic pain is a serious problem globally. Pain affects one in five adults, while an estimated one in ten suffers from chronic pain each year . BecauseAnacylcus pyrethrum (L.) Link (Asteraceae) is a native plant of North Africa  using appropriate models in mice. In addition, as inflammation is a process linked to oxidative stress and to the over-production of the reactive oxygen species (ROS), the antioxidant capacity of both extracts was also evaluated.Although some tivities , no scieAnacylcus pyrethrum (L.) Link (Asteraceae) was collected in June 2014, from Oukaïmeden (74 km from Marrakech) at 2,600 m of altitude in the High-Atlas Mountains (Morocco). The plant was identified by Professor A. Ouhammou, a taxonomist in the department of Biology, Faculty of Sciences Semlalia, Cadi Ayyad University. A voucher specimen was deposited at the Faculty’s Herbarium (Mark 8258).The roots were separated from the aerial parts of the plant and dried under shade. They were ground to a fine powder using a grinder apparatus. Root powder (1 g) was stirred with distilled water (20 ml) for 24 h at room temperature. The aqueous macerate was centrifuged (1200 rpm) for 15 min. The supernatant was lyophilized (yield = 20% w/w) then stored in a freezer at -20°C until experimental use. For methanol extract preparation, the powder of APR (400 g) was exhaustively extracted with methanol in a Soxhlet apparatus. The methanol extract was concentrated to dryness under vacuum. The residue (21.8% w/w) was stored at -20°C for several months, until the experimental use.Aqueous and methanol extracts of APR were screened for the presence of flavonoids, alkaloids, terpenoids, tannins and saponins. The qualitative determination of these phytochemicals was conducted using previously reported methods .1. Test for flavonoids: 2 ml of APR extract was evaporated and the residue was taken up in 5 ml of alcohol (50%) and 1 ml of concentrated hydrochloric acid. After addition of a few magnesium chips, the presence of flavonoids was indicated by the apparition of a red color.2. Test for terpenoids: 1 mg of APR extract was dissolved in a few drops of acetic acid in 3 ml of the mixture (acetic anhydride-concentrated sulfuric acid 50:1 v/v). The development of a green color indicated the presence of terpenoids.3.3; 9%). Test for tannins: APR extract (5 mg) was dissolved in 20 ml of distilled water and heated to boiling on a hot plate. Tannins were detected by the apparition of a green color after the addition of a few drops of an aqueous solution of ferric chloride (FeCl4. Test for saponins: APR extract (500 mg) was dissolved in 10 ml of distilled water in a test tube. The tube was shaken vigorously for 15 s and then allowed to stand for 15 min. The presence of stable foam indicated the presence of saponins.5.vacuum of the organic layer, the residue was dissolved in 1 ml of methanol. A few drops of Dragendorff reagent were added to the solution; the formation of a precipitate was taken as a hint for the presence of the alkaloids. Test for alkaloids: APR extract (500 mg) was stirred in 50 ml of sulfuric acid (0.1 N) for 15 min. After filtration, a concentrated ammonia solution (5 ml) was added to the solution. The alkaloids were then extracted with 50 ml of dichloromethane. After evaporation under in vivo bioassays. The animals were provided by the Animal Care Facility of the Faculty of Sciences Semlalia, Cadi Ayyad University, Marrakech, Morocco. The mice were kept under constant conditions of ambient temperature (22 ± 2°C) under a 12 h light/12 h dark cycle, with ad libitum access to food and water. All animal procedures were in strict accordance with the guidelines of the European Council Directive (EU2010/63). Care was taken to minimize the number of animals used for the experiments. All efforts were made to minimize any animal suffering, and the study met the ethical standards and approvals of the Council Committee of the research laboratories of the Faculty of Sciences, Cadi Ayyad University of Marrakech.Adult Swiss male mice (25–35 g) were used for Indomethacin was purchased from Laprophan (Morroco) and naloxone from Hospira (United States). Acetic acid, formalin, xylene, xylazine, ketamine and CFA  were obtained from Sigma–Aldrich (France).The acute toxicity study was conducted according to the Organization for Economic Cooperation and Development (OECD) guideline no. 423 , where tThe xylene-induced ear edema test was performed as previously described . BrieflyEight groups of animals (six mice per group) were assigned to this test. Mice were anesthetized with a mixture of ketamine (50 mg/kg) and xylazine (2 mg/kg) cocktail. All groups, except vehicle control group, received 20 μl of CFA by a subcutaneous injection in the plantar surface of the right hind paw . Twenty To measure the nociceptive reactivity to the application of mechanical stimuli to the hind pad, each mouse was placed in an individual observation cage (12 cm × 12 cm × 12 cm) with a mesh floor allowing access to the ventral surface of the hind pads. The animals were accustomed to the cage and the experiment room for at least 10–15 min or at the end of the exploratory behavior. Then mechanical hypersensitivity was assessed as described by To investigate the effect of chronic treatment on paw withdrawal, and the possible development of tolerance, mice  were treated with AEAPR, MEAPR , indomethacin (10 mg/kg), or vehicle (10 ml/kg) once a day for 5 days successively. In order to investigate the possible development of tolerance, the treatment was interrupted for 3 days (from day 6 to day 8) and reinitiated for 2 days again (day 9 and day 10). The mechanical hypersensitivity was assessed 3 h after each daily treatment . For the days 6–8 (without treatment), the test was performed at exactly the same time as the previous days.In this test, animals were individually placed on a hot plate with an adjustable temperature (to 55 ± 1°C) . The reaThis test was performed in mice, according to the method described by Formalin test was carried out as previously reported . Ten groTo evaluate any coordination disruption, non-specific muscle-relaxant or sedative effects of APR extracts, mice were subjected to the rotarod task and open-field test. The motor coordination of the mice was evaluated on the rotarod apparatus at a constant speed of 12 rotations per minute. Twenty-four hours prior the drug testing, animals were tested and those who remained at the rotating bar for the full 300 s during three consecutive trials were used for the subsequent experiments. The selected animals were randomly distributed into groups of six mice and received AEAPR or MEAPR orally at a dose of 125, 250, or 500 mg/kg. The control group received the same volume of vehicle (10 ml/kg). An additional group received indomethacin (10 mg/kg) and served as a positive control. Rotarod tests were performed prior to drug administration  and at 30, 60, and 120 min after administration. Latency to fall off was measured for each session (up to 300 s).In order to evaluate eventual motor impairment induced by plant extract, the mice were placed individually in an observation chamber 60 min after oral treatment with vehicle, indomethacin or APR extracts. The open field apparatus used was a 50 × 50 cm square arena with 30 cm high black walls. The animal was placed in the center of the arena and distance traveled was scored during 10 min, using the videotracking EthoVision XT8.5 software . The animal was returned to its home cage, and the apparatus cleaned with ethanol 70% to remove any odor.b – Aa)/Ab] × 100, where Ab is the absorbance of DPPH alone in methanol (control) and Aa is the absorbance of DPPH in the presence of the test substance. Quercetin and butylated-hydroxyl-toluene (BHT) were used as positive controls.The hydrogen or electron donation ability of APR extracts  was measured using the stable radical 1, 1diphenyl-2-picryl hydrazyl (DPPH) assay . Variousβ-caroteneafter2hassay/Ainitialβ-carotene) × 100, where Aβ-caroteneafter2hassay is the absorbance of β-carotene remaining in the sample after 2 h and Ainitialβ-carotene is the absorbance of β-carotene at the beginning of the experiment.The β-carotene/linoleic acid test evaluates the lipoperoxydation inhibitory effect of a compound or a mixture of compounds. The method described by +3 to Fe+2 was investigated using the method of 3 , and the absorbance was measured at 700 nm in a spectrophotometer. BHT and Quercetin were used as positive controls.The ability of APR extracts to reduce Fepost hoc analysis was used to examine the time-courses of mechanical, thermal and anti-edema effects after various treatments. Kruskal–Wallis ANOVA or one-way ANOVA followed by post hoc testing with the Student-Newman–Keuls test for multiple comparisons were used to measure variance of mouse behavior between groups. The significance of the differences between the means in the antioxidant activity was assessed by Student’s t-test. The significance threshold was set at p < 0.05.Statistical analysis was performed using SigmaPlot 11.0 software. All data were presented as mean ± SEM for six mice per group. A two-way analysis of variance (ANOVA) with repeated measures followed by Holm–Sidak The qualitative phytochemical screening of AEAPR and MEAPR showed the presence of alkaloids, flavonoids, tannins, saponins, and terpenoids in both extracts.p > 0.05) or in organ weights (p > 0.05) were observed at 14 days after AEAPR or MEAPR administration (Table 1).The oral administration of AEAPR or MEAPR at doses up to 5000 mg/kg did not produce any visible signs or symptoms of toxicity in mice. No mortality and no significant changes in body weights (p < 0.001). Indomethacin, as well as both APR extracts at tested doses, reduced xylene-induced ear edema compared to the vehicle-treated control group . Indeed, Kruskal–Wallis one-way analysis of variance confirmed a high significant difference between all groups . The post hoc analysis showed that the pretreated groups with APR extracts or indomethacin decreased significantly (p < 0.001) the edema induced by xylene in comparison with vehicle-treated group. In the group treated with indomethacin, the inhibition of ear edema induced by xylene was significantly higher than that of the group treated with 125 mg/kg of MEAPR or AEAPR , whereas the oral administration of the two extracts at a dose of 500 mg/kg exhibited a very strong anti-edema effect compared to the indomethacin group  . However, we observed no significant difference in ear edema between AEAPR and MEAPR at a dose of 250 mg/kg vs. indomethacin  nor between AEAPR vs. MEAPR groups . The positive control, indomethacin, inhibited edema by 49%, whereas MEAPR and AEAPR at a dose of 500 mg/kg inhibited it respectively by 65% and 62% compared to the group treated with the vehicle.Topical application of xylene caused a significant increase in the right ear section’s weight (11.68 ± 0.49 mg) when compared to the vehicle-treated left ear (5.22 ± 0.29 mg)  in paw volume after 24 h (from 4.50 ± 0.08 mm to 5.10 ± 0.05 mm). The paw thickness was reduced respectively by MEAPR, AEAPR and indomethacin after 30, 60, and 120 min of oral administration  . This reduction was statistically different between experimental groups , and also between different post-treatments’ times . Both APR extracts showed dose- and time-dependent anti-inflammatory effects. At higher doses (250 and 500 mg/kg), this effect became significant, starting from the third hour after AEAPR administration  and starting from the second hour for MEAPR . It does not become significant until the fourth and fifth hour after the administration of the low dose (125 mg/kg) of MEAPR or AEAPR . In addition, at a higher dose, AEAPR and MEAPR exhibited a greater reduction effect in paw thickness than indomethacin from time points 3 h , to 7 h .Complete Freund’s Adjuvant injection caused a significant increase , 24 h after CFA injection . The single oral administration of AEAPR or MEAPR, at 250 and 500 mg/kg, reduced the mechanical hypersensitivity induced by CFA. The paw withdrawal threshold increased significantly from point time 1 h 30 to 3 h and remained high up to 7 h . Two-way repeated measures ANOVA revealed that the withdrawal threshold was significantly different between groups  and varied significantly with time . The post-hoc analysis showed a significant increase of withdrawal threshold at time point of 1h30 in mice treated with AEAPR or MEAPR (250 and 500 mg/kg) and indomethacin compared to the CFA group . The CFA group did no differ at 125 mg/kg of both extracts, except for MEAPR at the time point 3 h . Moreover, no significant difference was observed between indomethacin and MEAPR or AEAPR at 500 mg/kg .The intraplantar injection of CFA produced noticeable mechanical hypersensitivity . The results demonstrated that the threshold responses of animals after AEAPR or MEAPR treatments were increased during the observation period compared to CFA group. When the treatments were interrupted for 3 days, mechanical allodynia was re-established. On the 9th day, the treatments were restarted and it was observed that both extracts of APR once again reduced mechanical allodynia.To investigate the effects of repeated treatment, mice received daily MEAPR or AEAPR , indomethacin, or vehicle for 5 days, interrupted for 3 days, then given daily for 2 more days  = 171.13, p < 0.001] and time . On the first day of treatment (day 1), except for the dose of 125 mg/kg of both extracts, the post hoc analysis showed significant differences between CFA group and MEAPR , AEAPR  and indomethacin  treated groups. In addition, there was no significant difference, neither between MEAPR and AEAPR groups at 500 mg/kg nor between MEAPR and indomethacin groups (p > 0.05), whereas a significant difference between AEAPR and indomethacin groups  was shown. During the first treatment period (from day 1 to day 5) with MEAPR, AEAPR and indomethacin, the withdrawal thresholds were significantly increased (p < 0.001) compared to the day before the treatment (day 0). The interruption of the treatment induced a reduction in paw withdrawal threshold of animals treated with APR extracts at all doses. However, this weakening of the antinociception reaction after the treatment interruption was not significant at the higher dose. The significant increase of threshold responses reappeared with the resumption of treatment .A two-way repeated measures ANOVA (treatment and day) revealed a significant effect of treatment . In fact, post hoc analysis showed that the extent of induced writhing at doses of 250 and 500 mg/kg of AEAPR or of MEAPR was significantly reduced compared to the vehicle group ; whereas both extracts of APR at 125 mg/kg, did not produce an antinociceptive effect (p > 0.05). In addition, the number of mouse abdominal constrictions induced by acetic acid in MEAPR treated group did not differ from that of the AEAPR treated group (p > 0.05) at high doses. Moreover, the test revealed that both extracts, at a dose of 125 mg/kg, reduced the number of writhes but to a lesser degree than that observed with indomethacin , unlike the effect of the two higher doses (250 and 500 mg/kg), which did not significantly differ from indomethacin’s one (p > 0.05). APR extracts treatments at 125, 250, and 500 mg / kg induced contraction inhibition of 19.2, 30.9, and 52.2% respectively for AEAPR and 22.3, 31.6, and 56.7%, respectively, for MEAPR, while the standard drug (indomethacin) had an inhibition rate of 46.4%.The oral administration of AEAPR or MEAPR at a dose of 250 and 500 mg/kg reduced the number of writhes compared to the vehicle group . This effect began 60 min after oral administration of high doses and then disappeared after 240 min. At a lower dose of APR extracts (125 mg/kg), the paw withdrawal latency started to increase significantly after 90 min of treatment with MEAPR and only after 120 min of treatment with AEAPR ; two-way repeated measures ANOVA (group and time effect) confirmed these findings . Compared to baseline, the post hoc analysis showed that the treatment with indomethacin, MEAPR or AEAPR at higher doses (250 and 500 mg/kg) increased significantly the paw withdrawal latency, starting from 60 to 180 min (p < 0.001). In addition, naloxone (an opioid antagonist), which showed any significant effect on thermal sensitivity , partially reversed the effect induced by AEAPR or MEAPR at 500 mg/kg in the hot plate test. In fact, we observed a decrease of the paw withdrawal latency of 33.68 or 38.77%, respectively, in comparison with both AEAPR and MEAPR at time point 120 min.In the hot plate test, the oral administration of indomethacin, MEAPR or AEAPR significantly increased the paw withdrawal latency compared to the vehicle-treated group , and one-way ANOVA confirmed these differences . The post hoc analysis showed a significant difference between treated groups with 250 and 500 mg/kg of MEAPR or AEAPR and vehicle-treated group (p < 0.05). In addition, the analysis showed a significant difference between AEAPR at 125 mg/kg and indomethacin (p < 0.05), whereas there was no significant difference between indomethacin and MEAPR treated groups (p > 0.05). Concerning the second phase, all treatments markedly reduced the licking time of the injected paw in comparison with the group treated with vehicle . The one-way ANOVA indicated that all treatments induced significant differences of the mean paw licking time in comparison to vehicle-treated group . The post hoc analysis showed that the licking times at all doses of each APR extract and indomethacin were significantly (p < 0.001) less than that of the vehicle group. In contrast to the first phase, the effect of MEAPR and AEAPR at 500 mg/kg scored higher (p < 0.05) than indomethacin in the second phase with percentages of inhibition of 66, 64, and 43%, respectively. The pre-treatment with naloxone (1 mg/kg) reversed the antinociceptive activity of AEAPR and MEAPR at a dose of 500 mg/kg, i.e., an increase in the licking time of the first phase  and the second phase  in the formalin test. While the effect of naloxone treatment alone, did not differ from that of the vehicle (p > 0.05).In the formalin test, pain responses such as licking of the right hind paw were expressed in the first and second phases. The licking time of the first phase was reduced when the mice were treated with 125, 250, and 500 mg/kg of MEAPR , did not significantly reduce locomotion in the open field , and produced no difference in latency to fall off the bar in the rotarod test .Anacylcus pyrethrum root was assessed by three complementary in vitro antioxidant assays: the DPPH, the FRAP and the BCB assays. The concentrations that led to 50% inhibition (IC50) are given in Table 2. Note that low IC50 values reflect better protective action. The results showed that both Anacylcus pyrethrum extracts (MEAPR and AEAPR) exhibited similar and interesting antioxidant activity, especially in DPPH test with IC50 values of 12.38 ± 0.28 μg/ml and 13.41 ± 0.67 μg/ml respectively. The antioxidant potencies of both extracts were significantly less than those of the reference antioxidants butylated hydroxytoluene (BHT) and quercetin (Table 2). No significant difference was observed between AEAPR and MEAPR in any of the three antioxidant assays (p > 0.05). Recorded results showed that among the used bio-assays, the antioxidant activities of MEAPR and AEAPR were significantly higher, with DPPH than with other assays (p < 0.001).The antioxidant activity of 50 higher than 5000 mg/kg. According to the chemical labeling and classification of acute systemic toxicity recommended by OECD, AEAPR and MEAPR were assigned to the lowest toxicity class  than that of indomethacin (49%). This model is widely used to evaluate anti-inflammatory topical steroids and non-steroidal anti-inflammatory agents, especially those that inhibit phospholipase A2 . In addiIn addition, to investigate the effects of APR in a sustained inflammation model, we evaluated the effect of AEAPR and MEAPR on inflammation induced by intraplantar injection of CFA. Our results showed for the first time that acute or chronic oral treatments of animals with MEAPR or AEAPR were effective in preventing not only paw edema caused by CFA injection, but also mechanical hypersensitivity. Moreover, the anti-edematogenic and antinociceptive actions of APR extracts were evident from an early stage (1 h30) and maintained up to 7 h.It has been reported that CFA induces persistent pain that results mainly from the involvement of macrophages and T lymphocytes in the injected rat paw, followed by a paw swelling and leukocyte infiltration of the synovium and surrounding tissue, which contribute to chronic inflammation and osteolytic lesions . In addiEchinacea purpurea roots exerted immuno-modulatory effects, especially with a decrease of plasma protein levels of certain pro-inflammatory cytokines (IL-8 and IL-6) and inversely an increased expression of anti-inflammatory molecules such as IL-10. Thus, although we didn’t quantify the levels of the cytokines, we may predict that APR extract may act via the same product  to blunt the induced inflammation.Since the antinociceptive effect of AEAPR and MEAPR is associated with anti-inflammatory action, its continuing antinociceptive effect in the chronic pain model may be due to a reduction in the cytokine and prostanoid release which reduced sensitization of the nociceptors. It should be noted that Assessment of AEAPR and MEAPR effects on the abdominal constrictions elicited by acetic acid showed a marked suppression of writhing response in the visceral pain model. This test is mainly used to screen antinociceptive activity . It has The central antinociceptive activity of MEAPR and AEAPR was evaluated in the hot plate test. This test is a widely used model for acute thermal nociception to evaluate specifically central nociception . ThroughTo discriminate between the peripheral and central antinociceptive effects of APR extracts, the formalin test was used. Our results revealed that both AEAPR and MEAPR have acted effectively in both phases of the formalin test. It is known that the intraplantar injection of formalin induces two phases of pain sensitivity . The firMoreover, in this study, we demonstrated that the antinociceptive action of AEAPR and MEAPR was partially antagonized by naloxone in the hot plate, and in the formalin test (first and the second phase). These results suggest that the APR extracts mechanism involves in part opioid receptors.In addition to this, we did not detect any disturbances in the locomotor activity or motor performance in animals treated by AEAPR or MEAPR up to 500 mg/kg. This suggests that both extracts at the highest effective dose, have no muscle-relaxant or central depressant action in models of nociception used in our study. Our result is in opposition of the study of Anacyclus pyrethrum root were investigated for their antioxidant action against free radicals using diverse methods . The results showed that APR extracts, according to DPPH method, have a strong scavenging activity, but with the capacity to protect against lipid peroxidation (BCB test). Indeed, Anacyclus pyrethrum effectively inhibits oxidative stress, which is in accordance with previous studies (Inflammation is a process that involves a series of phenomena that may be due to several agents. It is usually associated with pain which is a secondary phase resulting from the release of analgesic mediators . The inf studies .in vitro, are able to scavenge a wide range of ROS like superoxide and nitric oxide radicals (The antioxidant potential of APR extracts may be due to their phytochemical constituents. In fact, phytochemical screening has proved the occurrence of alkaloids, flavonoids, saponins, tannins, triterpenes and sterols. Detection of these phytochemicals in APR agrees with earlier findings . The pheradicals . In addiAnacyclus pyrethrum roots are non-toxic substances, with good central and peripheral antinociceptive effects, which is beneficial and researched in traditional medicine. The qualitative phytochemical analysis and the antioxidant activity in vitro have revealed the presence of several antioxidant phytoconstituants such as flavonoids, alkamides, saponins and tannins in Anacyclus pyrethrum root extracts.Our study demonstrated that aqueous and methanol extracts of Many mechanisms of specific action to those phytochemical compounds could be responsible for anti-inflammatory and antinociceptive activities observed in the present work. However, further studies are needed to isolate the pharmacologically active compounds and elucidate their exact molecular mechanism in the anti-inflammatory and antinociceptive process of APR.HM, MB, ZS, and SB designed the experiments; HM and OB performed the experiments, HM, SB, MB, and ACG performed the analysis of the data; HM And SB assembled the figures. HM, SB, MB, and ACG wrote and edited the manuscript. All authors validated it.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Evidence is emerging that the interaction between male seminal fluid and female tissues promotes fertility, pregnancy, and health of offspring. This includes the acceleration of ovulation in a species known as a spontaneous ovulator, the domestic pig. Earlier studies revealed that seminal plasma acts by a local mechanism in the female pig. The aim of the present study was to examine local short-term and mid-term effects of seminal plasma (SP) on mRNA expression of immunoregulatory genes and transcripts associated with follicle- and oocyte maturation. In the porcine animal model, effects on mRNA expression in the female tract and preovulatory follicles were examined. SP suppressed mRNA expression of Prostaglandin-Endoperoxide Synthase 2 (PTGS2) ipsilateral to the infused uterine horn which was associated with a lower presence of immune cells in the uterine epithelium and lower PTGS2 immunoreaction. Depending on the sampling time (2 h vs. 17 h) and hormonal status, SP altered significant correlative relations of mRNA expression between PTGS2 and the transcripts Tumor Necrosis Factor Alpha, Tumor Necrosis Factor Alpha-Induced Protein 6 and Pentraxin 3 in uterus, granulosa and cumulus cells. A modulatory effect of SP on the oocyte gene network comprising eight oocyte transcripts was observed: uterine exposure to SP induced positive correlations  of maturation promoting factors among each other and with cumulus cells on the side of the treated horn. In conclusion, SP orchestrates the gene network regulating the bidirectional communication between oocytes and surrounding somatic cells. The modulation of the immune-cytokine network of the female reproductive system could contribute to the previously reported SP-induced acceleration of ovulation in the porcine species. Although it seems a little surprising that in vivo insemination as commonly used in human and animal assisted reproduction can give satisfying results with only residual seminal plasma (SP), there is now no doubt that the interaction of SP components with the female reproductive tract enhances the likelihood of successful establishment of pregnancy. Exposure of the uterus to SP triggers endometrial cells and resident leukocytes to synthesize chemokines and other mediators . The While there has been substantial work on the uterine reaction to seminal fluid, until recently there has been only sparse information on ovarian responses. In a non-primate species also classified as a spontaneous ovulator, the domestic pig, seminal fluid contact accelerate ovulation , possiblBeyond this background, we expand previous studies on the local effect using a porcine animal model allowing the unicornually application of SP whilst the contralateral horn served as control. The unique benefit of this model is having an animal as essentially its own control even though a systemic effect additionally to the previously described predominant local SP-effect ,11 cannoUnless otherwise stated all chemicals were obtained from Sigma .Sixteen hybrid gilts, aged 7–13 months, weighing between 140 and 180 kg were used in this study. All animals had experienced at least one spontaneous cycle prior to the experiment. They were housed in groups of two to three on straw in covered barns at the Unit of Reproductive Medicine, University of Veterinary Medicine Hannover. The gilts were fed a standard commercial diet of pellets and given free access to drinking water. The study was approved by the independent ethics committee of the Lower Saxony Federal State Office for Consumer Protection and Food Safety, Oldenburg, Germany (research permit number 33.12-42502-04-07/1261).th day onwards, the ovaries were examined by transcutaneous sonography  once daily, from the 18th day on four times daily  to determine the follicle size.Studies were performed during spontaneous oestrus. Oestrus detection started at day 16 p.ov. by visual examination of the vulva and backpressure test in the presence of four different boars at intervals of four to six hours. The beginning of oestrus was set at the middle of the time interval between the last rejection and the first tolerance. From the 16Semen samples of ten mature boars with known fertility were collected over two months. The single ejaculates were centrifuged three times at 3.370 x g for ten minutes and the cell free plasma was stored frozen at -27 °C. The plasma pool was prepared by thawing the frozen samples, mixing and aliquoting in 100 ml bottles. Bottles were stored at -27 °C until use.® sol. 1 vol%, Eifelfango, Bad Neuenahr; 0.02 mg/kg KGW i.m.) and a combination of ketamine–/ azaperone  were administered for premedication. Approximately 30 min later, anaesthesia was progressively deeper obtained by intravenous injection of ketamine (Ursotamin®) via an indwelling ear vein catheter. After endotracheal intubation, anaesthesia was maintained using a mixture of isoflurane, nitrous oxide, and oxygen. Throughout surgery, animals received an intravenous infusion of electrolytes  and fundamental physiological variables were recorded using an anaesthesia monitor .Uterine infusion of SP and phosphate buffered saline (PBS) were performed between 3 and 15 h after the calculated onset of oestrus under general anaesthesia. An intramuscular injection of atropine  were positioned around each uterine horn approximately 10 cm distal to the uterine body. 100 ml of the 37 °C warm SP were slowly injected into one uterus horn and 100 ml 37 °C warm PBS into the contralateral horn using disposable sterile syringes, polyethylene extension and needle. An ovario-hysterectomy and a partial hysterectomy of 40 cm of the cranial uterine horns including the uterotubal junction was performed after 2 h  during the same surgical approach or after 17 h  at a second surgery. The mid-ventral incision was closed with three separate layers. Animal were returned to straw-bedded recovery boxes and received analgesic  and antibiotic  treatment.Following aseptic procedures, the reproductive tract was exposed via a mid-ventral incision and, with the minimum handling of tissues, the number of mature pre-ovulatory Graafian follicles was recorded for each ovary. Double ligatures of an absorbable filament  immediately before treatment and immediately before ovariohysterectomy. Immediately after collection, blood samples were centrifuged at 3000 x g for 10 minutes and the plasma was stored at -27 °C until hormone analysis. Progesterone was measured by means of the competitive chemiluminescence-enzyme-immunoassay IMMULITE® system  according to the manufacturer’s instructions. The intra-assay coefficient of variance was 6.3%. Oestradiol was measured with an enzyme-immunoassay as described earlier , 100 mg BSA). 350 μl PBS was added and samples were centrifuged at 1000 x g for 10 min. Afterwards, the supernatant was discarded and the cell pellet was frozen at -80 °C.Immediately after ovario-ectomy, ovaries were placed in 37 °C warm PBS supplemented with 1% BSA and were transferred to the lab. Within 5 min after collection, granulosa cells, cumulus cells, and oocytes were separately collected from Graafian follicles with diameters > 5 mm: follicles were punctured individually with an insulin syringe and a small needle (22 G). The aspirated fluid was transferred into Tissue Culture Medium 199 (TCM 199) and granulosa cells and COC were isolated under a stereo microscope using two 22 G needles. The oocytes with residual cumulus cells were denudated with a stripper-pipette  in several steps using three pipette tips (Mid Atlantic Diagnostics) of different diameter . To ensure a good oocyte quality, denudation had to be finished within 30 minutes. Denudated oocytes were washed three times in 37 °C PBS with 1% PVA, transferred with a maximum of 1.5 μl PBS + PVA into a RNase and DNase free Eppendorf cup  and stored at -80 °C until analyses. In parallel with oocyte preparation, isolated granulosa and isolated cumulus cells of single follicles/oocytes were transferred into 50 μl TCM Air , 70% alcohol (2 min) and a rinsing step in PBS (3 x 5 min). Then a pretreatment with TEC-buffer  was performed for PTGS2. Antibody MAC387 required treatment with Proteinase K solution . For all antibodies this was followed by rinsing in PBS (5 min) and incubation with 20% normal goat serum for 20 min. The primary antibody was diluted with PBS (+1% BSA) to a concentration determined in advance  and incubated in a moist chamber at 4°C overnight. After rinsing the sections with PBS (3 x 5 min) they were incubated with secondary antibodies (goat-anti-mouse or goat-anti-rabbit 1:200 in PBS for 45 min at RT in a moist chamber). Another rinsing step in PBS followed (3 x 5min). Then the sections were subjected to the ABC system (ABC Vectastain Elite Kit) for 30 min at RT in a moist chamber and rinsed again in PBS (3x5min) before they were treated with the chromogen . After stopping the colour development by a rinsing step in PBS (1 x 5min) and running tap water (10 min), the sections were counterstained with hemalum for 5 sec. Finally, the sections were rinsed in running tap water for 15 min and dehydrated in graded alcohols . They were mounted with Eukitt® after immersion in Xylol (2 x 5min). Negative controls were performed by replacing primary antibodies with either PBS or rabbit or mouse IgG which were diluted like the primary antibodies.Sections of 3–4 μm were produced from the paraffin embedded tissue and subjected to routine haematoxylin-eosin staining for general evaluation. Additionally, immunohistochemistry was performed with antibodies against neutrophil granulocytes and macrophages  and PTGS2 . Briefly, sections were deparaffinized in xylol (2 x 10 min) and then rehydrated in a series of alcohols . This was followed by incubation in a HFrom each animal one section from each localization was analysed in a blinded fashion.Evaluation of the immunoreaction of the MAC387 antibody was conducted by light microscopy (Leica LMD 7000) at a magnification of x400 in two ways. For both, ten fields of view per section were analysed. For the locations subepithelial stroma, stratum compactum and reticulare, endometrial and perimetrial blood vessels, myometrium and perimetrium the amount of cells was assessed semiquantitatively  because counting was impossible due to high cell numbers.For the PTGS2 antibody, in ten fields of view the staining intensity was classified in four categories (negative (= 0), weak reaction (= 1), positive (= 2) and strongly positive (= 3)) for the locations uterine epithelium, stratum compactum and reticulare, glandular epithelium, endometrial blood vessels and myometrial and perimetrial immune cells.PTGS2, Interleukin 6 (IL6), Pentraxin 3 (PTX3), Peroxisome Proliferator-Activated Receptor-Gamma 1 (PPARG1), Tumor Necrosis Factor Alpha (TNFA), Tumor Necrosis Factor Alpha-Induced Protein 6 (TNFAIP6) and Ubiquitin B (UBB) was analysed in epithelium of the uterine horn, the utero-tubal junction, granulosa cells and cumulus cells. To isolate RNA from the laser microdissected epithelium and the granulosa and cumulus cells stored at -80 °C, lysis buffer was added to the samples and RNA was extracted with the RNeasy® plus micro Kit (Qiagen) according to the manufactures instructions. To ensure RNA quality and quantity, every single RNA sample was analysed with the Experion® RNA Standard Sense Chips and the Experion® RNA Standard Sense Analysis Kit®  in the Experion® Automated Electrophoresis System® . To obtain same amounts of cDNA from each sample, RNA was diluted to 15 ng of total RNA/μl (epithelium of uterus and UTJ), 17 ng of total RNA/μl (granulosa cells) or 4.5 ng of total RNA/μl (cumulus cells).The mRNA expression of six immunoregulatory transcripts, ™ Recombinant RNase Inhibitor and the reverse transcriptase Superscript® III  were added. The RT reaction was carried out for 5 min at 25 °C, 60 min at 50 °C and 70 °C for 15 min according to manufacturer’s instructions.Immediately thereafter, cDNA was generated by adding random primers and dnTPs , water was added to a total volume of 13 μl. After incubation at 65 °C for 5 min, 5x First-Strand Buffer, 0.1 DTT, RNaseOUT6−100 copies) of the respective cDNA sub clones. Subsequently a melting curve was performed starting at 60 °C increasing the temperature every 15 sec for 0.3 °C to verify the amplified fragments.Messenger RNA copies were quantified with a Step One Plus real time PCR system . One microliter of the cDNA preparation representing 15 ng , 17 ng (granulosa cells) or 4.5 ng (cumulus cells) were distributed to individual wells. Gene specific primer and the SYBR Green PCR Master Mix  were added. Primer sequences, product sizes (bp) and accession numbers are available . Water wc-Mos, CCNB1, Mitogen-Activated Protein Kinase 1 (MAPK1), Cyclin-dependent Kinase 1 (CDK1), Proliferating Cell Nuclear Antigen (PCNA), Growth/Differentiation Factor 9 (GDF9), Bone Morphogenetic Protein 15 (BMP15) and Zygote Arrest 1 (ZAR1), were analysed in denuded and cryopreserved oocytes. Poly (A)+RNA was extracted as described by Stinshoff et al. [® mRNA DIRECT™ Micro Kit  and directly used for reverse transcription (RT). RT was carried out in a total volume of 20 μl. The reaction mix contained 1x PCR Buffer , 5 mM MgCl2 , 1 mM dNTPs , 2.5 μM random hexamers , 20 U RNase inhibitor and 50 U murine leukaemia virus (MuLV) reverse transcriptase . The RT reaction was carried out at 25 °C for 10 min, 42 °C for 60 min followed by denaturation at 99 °C for 5 min and was then stored on ice.Prior to RNA isolation, 1 pg of rabbit globin RNA was added as external control. Eight developmentally important transcripts, f et al.  with the2, SYBR® Green, stabilizer, fluoreszin), 0.2 μM primer forward and reverse each for the gene of interest and the external reference gene . The PCR reaction mix contained 10 μl Mastermix . A gene was only considered for statistical analysis if its mRNA expression levels were above detection level (> 100 copies) in samples from both sides of the genital tract in more than 50% of the animals c.f. . Data weThe mRNA copy numbers in samples from the uterus, the utero-tubal junction, granulosa cells, and cumulus cells showed high variations between animals for any given gene. In order to reduce the bias of the high inter-animal variations and achieve a more standardized comparison, coefficients of relative differences in mRNA expression were calculated, when testing for differences in mRNA levels between e.g. the SP-treated and PBS-treated side or between locations within the SP-treated or PBS-treated side of the genital tract.An exemplary calculation is given in The coefficient from the example is used for comparing the mRNA expression level at a given location (e.g. uterus) between the SP-treated side and the PBS-treated side of the genital tract. The coefficients were entered into the calculations for the statistical tests (Student’s t-test and Wilcoxon’s signed rank test) instead of the commonly used differences (deltas) in mRNA copy numbers. Both, Student’s t-test and Wilcoxon’s signed rank test are testing the hypothesis that no significant deviation from zero exists for a given set of numbers. If the hypothesis was accepted, no significant difference in mRNA expression levels was assumed. If the hypothesis was rejected, a significant difference in mRNA expression levels was assumed. Data are reported as original mRNA copy numbers in all tables and figures.Correlations between mRNA copy numbers of selected transcripts within a location or between locations were calculated with Spearman’s rank correlation coefficient (PROC CORR). Data of neutrophils in the uterine epithelium were analysed using Wilcoxon signed-rank test .Differences were considered statistically significant if P < 0.05.All animals exhibited signs of oestrus including hyperaemic, oedematous vulvae and had normally developed reproductive tracts. Ovarian follicles ranged between 7 and 11 mm in diameter and residual corpora lutea were present from the previous cycle. The median of the relation between oestradiol and progesterone at the time of treatment was 15.9 . In Group 2, the hormonal status was additionally examined at the time of tissue sampling, i.e. 17 h after treatment: the median of oestradiol and progesterone was 11.5 (range: 9.2 to 20.8).The number of neutrophils in the uterine epithelium was statistically significantly lower (p< 0.05) in the SP treated side when compared to PBS infused sides . SemiquaPTGS2 immunoreaction was higher in luminal epithelium of uterine horns compared to glandular epithelium . AdditioIL6, TNFA and TNFAIP, of the receptor proteins PPARG1 and PTX 3, and of the enzyme PTGS2 and of UBB as reference at the different location of the female tract both on treated and control sides is shown in PTGS2, TNFAIP6 and UBB was consistently expressed in all tissue samples in most animals. IL6 mRNA expression was found in the majority of gilts in the uterine epithelium in both groups of gilts and in granulosa cells of most animals in Group 2. No gene expression of IL 6 was detected in cumulus cells. Messenger RNA of TNFA was expressed in both epithelium of uterine horns and UTJ, but not in granulosa and cumulus cells. PPARG1 mRNA was expressed in all animals in granulosa cells only. Expression of the transcript PTX3 was consistently found in granulosa and cumulus cells, but only irregularly in uterine samples. Gene expression levels varied between animals. In Group 1 (sampling 2 h after treatment), expression levels in granulosa cells were related to the hormonal status  had significantly lower oestradiol/progesterone ratios compared to animals with low expression of these transcripts . Time or type of treatment did not influence the distribution of gene expression in the different tissue samples  of the cytokine genes l status . AnimalsPTGS2 in the epithelial cell layer of uterine horns treated with SP compared to control horns  in the epithelium of uterine horns compared to the uterine part of the UTJ. Differences were observed on both treated and control sides. Data are presented for animals in Group 1 in UBB did not differ between the two locations.In both groups of gilts, mRNA expression of PTGS2 , TNFA  and TNFAIP6  on the sides treated with SP, but not on contralateral sides. At 17 h after treatment (Group 2), no significant correlations were found.At 2 h after treatment (Group 1), mRNA expression was highly correlated between uterine horns and adjacent UTJ for the transcripts TNFA in the UTJ correlated significantly with expression of PTGS2  and PTX3  in granulosa cells ipsilateral to uterine horns treated with SP, but not on control sides  and cumulus cells  on both sides of the female tract. Data for both groups of gilts are presented in In animals of Group 1, gene expression of ol sides . Such coPTGS2 in cumulus cells and that of three oocyte transcripts PCNA, GDF9 and BMP15 as well as between PTX3 mRNA expression in cumulus cells and six oocyte transcripts PCNA, GDF9 and BMP15, c-Mos and CCNB1, were found on the sides of SP-treated uterine horns  between treated and control sides for all genes of interest, except for ZAR1. The relative mRNA expression levels did not differ between treated and control sides . At 2 h ne horns ; no corrnscripts . No signMAPK1 were positively correlated with mRNA–expression of CDK1, BMP 15 and ZAR1, and relative expression levels of c-Mos were positively correlated to those of CCNB1, PCNA, CDK1 .At 2 h after treatment (Group 1), relative mRNA expression levels of most transcripts were highly correlated  to each other on ovaries from both control and treated sides. Only on the side of the treated uterine horn, relative expression levels of NA, CDK1 . At 17 hNA, CDK1 . On the GDF9 and c-Mos as key factors showing most correlative relations with other transcripts. At 2 h after treatment, the interaction network between the transcripts of the eight studied oocyte factors seems to be denser regardless of the treatment. At this early time, the modulatory influence of SP induces positive correlations of maturation promoting factors including MAPK1 among each other and with other oocyte factors. The presence of high positive correlation of mRNA expression of the two oocyte-secreted growth factors GDF9 and BMP15 with mRNA molecule concentrations of both PTGS2 and PTX3 in cumulus cells only on the side of SP-treated uterine horns indicates that SP modulates the interaction of the different cell types in antral follicles. There is strong evidence that fine gene dosage mechanisms involving GDF9 and BMP15 play an important role in the regulation of ovulation across mammalian species including human [BMP15, GDF9, CCNB1, CDK1, PCNA and low expression levels of TNAIP6 in granulosa cells indicate an advanced oestrous stage approaching ovulation. The observed unilateral negative correlation between TNFAIP6 in granulosa cells and maturation associated oocyte transcripts at 17 h after treatment indicates that SP advances the synchronization of mRNA expression between the different cell types in the preovulatory follicle. Thus, we suggest that SP facilitates ovulation by influencing the oocyte-CC regulatory loop, at least in porcine species where large amounts of SP enter the female tract and spontaneous ovulation would only occur in the last third of the oestrus period. The effect of male genital fluids will inevitably enhance fertilization chances by preventing loss of sperm quality associated with ageing in the female tract. Whether exposure to SP also would affect oocyte quality is as yet unknown. The observation that SP induces a correlative relationship between PTX3 in cumulus cells and mRNA expression of regulators of oocyte maturation c-Mos, CCNB1 CDK1 in addition to BMP15 and GDF9 supports this hypothesis because these maternal transcripts as well as relative abundance of PTX3 mRNA in cumulus cells are considered as markers for development competence of female germ cells in human, pig and mouse [The identification of relationship among genes, proteins or molecules in biological pathways is critical for understanding complex biological activities and biological functions . The preng human ,27. In tnd mouse ,29.PTX3 mRNA in granulosa cells was ipsilateral highly positively correlated with mRNA expression of PTGS2 in cumulus cells. Cumulus cells similar to granulosa cells express numerous immune cell-related genes and are very sensitive to changes in the immediate environment [PTGS2, PTX3 and TNFAIP6 in granulosa cells were associated with different oestradiol/progesterone ratios thus explaining the inter-individual variations of mRNA expression. Animals with low oestradiol/progesterone ratios indicating an oestrus stage closer to ovulation exhibited higher expression of PTGS2 mRNA. This result corresponds with kinetic changes of pre-ovulatory gene expression in granulosa and cumulus cells of sows primed with hCG [At 2 h after single uterine horn infusion of SP, expression of ironment . Since cironment . Systemiwith hCG .PTGS2 mRNA is predominantly controlled by LH and steroid hormones [PTGS2 in the uterine epithelium was observed at 17 h after SP infusion compared to control which could be explained by the concomitant lower number of neutrophils. Resident immune cell and their interaction with epithelial cells are most likely to mediate local immune effects. In line with the present results, it was reported that porcine SP inhibits neutrophil immigration, accelerates PMN clearance from the uterus and suppresses chemotaxis of PMN Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


Homologous recombination (HR) is a complex biological process and is central to meiosis and for repair of DNA double-strand breaks. Although the HR process has been the subject of intensive study for more than three decades, the complex protein–protein and protein–DNA interactions during HR present a significant challenge for determining the molecular mechanism(s) of the process. This knowledge gap is largely because of the dynamic interactions between HR proteins and DNA which is difficult to capture by routine biochemical or structural biology methods. In recent years, single-molecule fluorescence microscopy has been a popular method in the field of HR to visualize these complex and dynamic interactions at high spatiotemporal resolution, revealing mechanistic insights of the process. In this review, we describe recent efforts that employ single-molecule fluorescence microscopy to investigate protein–protein and protein–DNA interactions operating on three key DNA-substrates: single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and four-way DNA called Holliday junction (HJ). We also outline the technological advances and several key insights revealed by these studies in terms of protein assembly on these DNA substrates and highlight the foreseeable promise of single-molecule fluorescence microscopy in advancing our understanding of homologous recombination. Homologous recombination (HR) is a well-conserved and essential biological process for both genetic exchange and the repair of double-stranded DNA (dsDNA) breaks ,2,3,4. TRepairing dsDNA breaks involves three phases—pre-synaptic, synaptic, and post-synaptic—corresponding to the trimming of the dsDNA backbone creating single-stranded DNA (ssDNA) overhangs, formation of a homologous exchange intermediate called a Holliday junction (HJ), and, finally, the resolution of the junction back into dsDNA ,7. A numThe groundwork towards unraveling complex pathways of the HR process have been laid out initially through a number of biochemical studies. The protein–DNA complexes that form during the HR process have been revisited more recently via single-molecule approaches and have significantly improved our understanding of the process ,23,24,25Various protein systems that are involved in the repair of dsDNA breaks via the HR pathway have several members, some of which act on both the dsDNA and ssDNA substrates. For instance, RecBCD is a complex of three enzymes which cuts and separates an ssDNA from genomic dsDNA at the chi sequence, leaving in its wake a trail of RecA protein bound to the ssDNA. The creation, protection, and repairing of the ssDNA segments is essential in the dsDNA break repair pathway. Typically, the ssDNA segments are protected by single-stranded DNA binding proteins (SSBs), where the SSBs wrap around the ssDNA portions to protect them from other cellular processes  and faciThe ssDNA-binding HR proteins have largely been explored using single-molecule FRET (smFRET) or DNA curtain platforms. The DNA constructs used in smFRET studies generally follow a simple design as shown in Single-strand DNA binding proteins (SSBs) and their analogs are essential in protecting ssDNA regions in various biological processes such as preventing the formation of ssDNA secondary structures and assisting in recruiting certain RecA mediators . The disThe properties and functions of RecA filaments have been the subject of major interest for single-molecule fluorescence studies in various stages of the HR process . In 2012The Rad51 nucleation was studied using an ssDNA construct with a FRET pair on the single-stranded region by Lu et al. . This stIn addition, the ssDNA platform has also been used to study other HR proteins. For example, in 2017, De Tullio et al.  used ssDInvestigation of dsDNA-binding HR proteins has been an area of considerable work in the fluorescence microscopy field. These proteins are critical to the presynaptic and synaptic stages of HR and often work in concert. Therefore, bulk methods have difficulty analyzing these complex interactions. Studies investigating and visualizing the binding of these HR proteins are by large performed on some linearized piece of dsDNA molecules hold in place under a fluorescence microscope. The mechanism by which the dsDNA molecules are held in place can be a “DNA curtain” approach, where the dsDNA molecules are bound to a functionalized slide surface on one end with continuous buffer flow to hold the DNA linear or are bound on both ends. Another method for holding a dsDNA molecule linear is binding the DNA to a functionalized polystyrene bead which is then held in a moveable optical trap, using a flow to keep the DNA extended. Exposure of DNA to different buffers is facilitated by physically moving the bead into various areas of a flow cell with buffers separated by laminar flow.The RecA filament formation was studied in 2006 by Galletto et al.  using ds−1 until this protein complex dissociates [In 2001, Bianco et al.  publishesociates . This wosociates . In 2012sociates  studied sociates . In thissociates . In 2013sociates  used optsociates .The essential HR protein, Rad51’s, filament-forming behavior was studied in 2006 by Prasad et al.  using a −1 [The double-bound ssDNA curtain  provided−1 .Studying proteins that bind to the HJ using single-molecule microscopy presents interesting challenges and opportunities due to the inherent conformational changes that occur in the HJ ,65,66. T2+-rich buffer to determine the energy landscape which governs the switching of the HJ between its stacked conformers. They concluded that the conformational switch must occur during many intermediate stages of HR [2+ increased, the HJ switched to its stacked conformation more often, stalling the branch migration [Single-molecule studies of the HJ have determined the structural dynamics of the HJ, particularly the shape that the junction adopts and the circumstances (primarily ionic environment and nucleotide composition at the core of the junction) under which it would switch from one stacked conformer to another. In 2003, McKiney et al.  used an es of HR . The stees of HR  in 2005.igration . The religration . They aligration . The conigration  using a igration . The binigration ,71 in 20igration ,71.2+ and Na+ showed a strong relationship between the ion concentration and RuvA binding activity (2+, suggesting that the RuvA–HJ interaction is primarily electrostatic in nature [Overall, the direct visualization of HJ-binding proteins is an ongoing research effort with only a few HR proteins having been studied. In 2015, Iwasa et al.  investigactivity B, with an nature .In 2019, Zhou et al.  used FREIn this review, we discussed recent work in the area of homologous recombination focusing on single-molecule fluorescence microscopy which has propelled our understanding of how HR proteins repair DNA breaks. More specifically, we described recent efforts to visualize how HR proteins operate on various DNA substrates—ssDNA, dsDNA, and Holliday junctions. We provided an overview of the methods used in these studies including FRET-based single-stranded DNA constructs, both single- and double-stranded DNA curtains, optical tweezers, and FRET-based analysis of Holliday junction analogs. These studies provided insights into the mechanism of protein binding, determined the kinetics of filament formation by various ssDNA-binding proteins, and revealed regulatory effects of protein–protein interactions on filament formation. More recent studies on HJ-binding proteins have revealed binding modes and effects of ionic environments on the binding interaction. These studies, along with bulk biochemical studies, have also started to decipher how these HR proteins contribute to the formation of branch migration complex and HJ-resolving machinery ,74,75,76In summary, using fluorescence microscopy, significant progress has been made in understanding the roles individual HR proteins play in the overall HR process. Some inroads have been made in determining the exact roles the HR proteins play on various stages of HR. However, an understanding of the synergistic effect that multiple HR proteins have on the HR process has not yet been fully achieved. Again, more work needs to be done to characterize the way different HR proteins work together to execute the entire HR process. Particularly, the steps and proteins involved in the transition from the synaptic to post-synaptic phases in human HR need more attention. To address these knowledge gaps, several labs around the world are using fluorescence microscopy and other forms of single-molecule techniques to dissect the HR process in detail. Combining the bulk biochemical work along with the molecular-level mechanistic insights obtained from single molecule work, we envision that we will have a complete picture of HR in the near future. Once this complete picture is established, it will significantly enhance our understanding of the HR process in the context of DNA break repair, and, in the long-term, it will become possible to intelligently target HR proteins for therapies.