Post Stroke Cognitive Impairments (PSCI) occur frequently in stroke survivors resulting in devastating consequences affecting daily living of survivors. Currently, there exists scarcity of sound assessment tools for the evaluation of PSCI as most of the commonly used scales offer a number of clinical (requires motor and linguistic skills) and cultural limitations (requires academic exposure). Therefore, current study was aimed at evaluating the efficacy of Neuro-Cognitive Assessment Battery for Stroke patients (N-CABS) in Pakistan.The participants were randomized in two groups including clinical and non-clinical group. N-CABS was administered on 61 clinical and 60 non-clinical participants . Both groups were matched on all demographic variables including; age, gender, education and occupation. The psychometric properties of N-CABS were established through a number of robust measures of validity  and reliability .***p<0.001). The findings of concurrent validity recommended a significant correlation between N-CABS and Montreal Cognitive Assessment (MoCA) . Similarly, discriminant validity also revealed significant group differences to exist on N-CABS (***p<0.001) suggesting N-CABS be able to discriminate between patients and healthy controls.The factor analysis suggested a two-factor solution  for N-CABS explaining 67% of total variance. A significant test-retest reliability was found (α = 0.92, The results of the current study favor N-CABS as a psychometrically strong screening instrument to screen PSCI in our culture. In only a few years, stroke has appeared as a standalone core Non-Communicable Disease (NCD) and gained recognition as having a larger influence on global health, which was previously rooted under the term of “cardiovascular diseases”.3The term stroke related disability is not only limited to physical health but it also covers the major aspect of mental health as well, that is cognition.6The assessment of PSCI is therefore crucial which not only plays its role in the process of diagnosis but neuropsychological evaluations are utilized to develop an effective treatment and rehabilitation plan.8Though there are many widely used neuropsychological screening tools for assessing executive functions in clinical practice today, however, most of these instruments were not developed with the purpose of assessing individuals with language and motor complexities.Therefore, the purpose of the study was to develop an instrument to address these issues and most importantly to enhance the efficiency and sensitivity of tool with respect to our culture. N-CABS was designed while keeping in consideration the socio demographic variables and possible motor and language difficulties among stroke patients. The difficulty level of tasks was intended to keep in accordance with educational level of Pakistani individuals to make tool sensitive in assessing PSCI. Furthermore, another purpose was to give equal weightage to linguistic and non-linguistics task unlike traditional neuropsychological test batteries. In tis study the goals were (1) to develop N-CABS (2) to identify cut points (3) to compare the performance of N-CABS with MoCA (most widely used tool to assess PSCI) and (4) to validate N-CBAS in an independent sample against a reference standard for cognitive impairment.The current neuropsychological test battery was developed following four main phases including; Phase 1 (Identification of the types of cognitive impairments after stroke), Phase 2 (Selection and collation of subtests assessing specified PSCI), Phase-3  and Phase-4 (Main study was carried out to assess the pattern of PSCI among patients and to develop psychometric properties).The most initial phase of research focuses on the determination of types of PSCI which involved review of the existing literature concerning PSCI,After the selection of subtests a try out phase was conducted to shortlist items from each complex subtest  on the basis of difficulty level. In order to calculate the difficulty level of items of subtests full subtests were administered on 30 participants. Furthermore, during this phase Naming subtest and Similarities subtest were also developed by selecting three most culturally common animals’ pictures as suggested by a sample of 30 participants during the phase of pilot study. The collated version of battery was then sent for expert validation to three of the professionals having expertise in neurological testing. All corrections were incorporated as suggested by the experts before moving to the main phase of study. Following these phases the neuro-cognitive test battery was finalized.The current study involved 121 participants divided into clinical (stroke patients) and non-clinical groups. The clinical group comprised of 61 participants whereas 60 participants were recruited in non-clinical group. The patients with unilateral stroke were recruited from the stroke ward in Neurology Departments of various government (n=4) and private hospitals (n=6) using purposive sampling. Inclusion criteria of the selection of participants in clinical group include; having a first unilateral stroke, age over 30 years, minimum education till primary and adequate post-stroke fluency in Urdu. Exclusion criteria include bilateral hemisphere, convergent conditions and disorders that might affect any cognitive skills under study, traumatic brain injury, infratentorial lesions, or patients with right hemispheric lesions who were assessed to have aphasia (crossed aphasia).The healthy matched controls were mostly relatives of patients or employees working in institutions where data was collected. All healthy participants were initially examined by physician for general health and only those participants were included having no motor, intellectual, neurological or sensory deficits or psychiatric disorders. Both groups were matched on demographic variables of age (over 30 years), education (primary or above) and occupation.The subtests selected for the purpose of screening of PSCI were taken from standardized batteries Wechsler Intelligence Scale for Children,The study was conducted after getting approval from the ethical committee of Institute of Clinical Psychology. The researcher took verbal informed consent by participants and provided them sufficient information regarding the purpose of the current study. The researcher also ensured the confidentiality of each participant and informed them about their right of leaving the study at any time they want.The data was analyzed using Statistical Package for Social Science, 21 version (SPSS 21). Analyses comprised of exploring descriptive information through descriptive statistics, exploratory factor analyses (EFA) to establish psychometric properties. The independent sample t-test was used to explore group differences whereas Pearson- product correlation was employed for assessing existing correlation between variables.The participants of the study involved both clinical and non-clinical individuals who were matched on various demographic variables  whereas the manipulated variable of interest was the presence of stroke which distinguished both groups. The frequency distribution of all significant socio demographic variables including age, gender, education, and occupation are provided in 2 (36) = 250.20, p < .005) strongly suggesting EFA. The analysis of diagonals of the anti-image correlation matrix support the inclusion of all 9 items in factor analysis as values of items found to be above 0.5. Furthermore, the output of communalities revealed the values of communality of all variables to be higher than 0.4, further confirming common variance shared by all items. All these indicators highly recommended EFA to be conducted on all 9 items.To validate NCABS Construct validity was opted which was done using Exploratory Factor Analysis (EFA) through Principal Component Factor Analysis (PCFA). Initially, in order to determine factors of N-CABS, the adequacy of EFA through PCFA had been assessed through Kaiser-Meyer-Olkin (KMO) measure of sampling adequacy and Bartlett’s Test of Sphercity. The value of KMO was 0.79 which was higher than recommended value of 0.6 whereas Bartlett’s Test of Sphercity was also significant (χ2 (28) = 234.22, p < 0.005).The initial Eigenvalues as shown in scree plot  obtainedFurthermore, a descriptive label was given to each respective factor based on the resemblance among subtests categorize under these two factors. The first factor being loaded with five subtests requiring verbal abilities including Attention, Paired Associate Learning Test (PALT), Naming, Reasoning and Arithmetic was named as Verbal Cognitive Abilities (Ver.CA). On the other hand, the second factor being loaded with three subtests involving visual abilities including Bell’s Test, Block Design and Non-Verbal Learning Test (NVLT) was named as Visual Cognitive Abilities (Vis.CA).***p<0.001) between both administrations suggesting N-CABS a reliable measure. The internal consistency was established through inter item correlation, the findings of which are presented in Other psychometric properties of N-CABS were established using reliability measures  and validity measures . The test retest reliability was determined by re-administering N-CABS on 10 patients after duration of one week. The Cronbach alpha of N-CABS found out to be 0.92 with a robust correlation (***p<0.001) between both measures suggesting N-CABS a valid measure for assessing cognitive impairments. Moreover, discriminant validity was conducted using known group validity to examine how well NCABS discriminates between group of patients and healthy controls. In order to assess group differences on N-CABS independent sample t-test was carried out. The figures in the p value across all the subtests and total N-CABS found out to be greater than 0.001*** alpha level. Therefore, the findings suggest a substantial presence of PSCI among patients of stroke and also N-CABS was able to successfully discriminate those clinical group and non-clinical group on the basis of participants’ performances.Further validation of N-CABS was done using two measures of validity, including; concurrent validity and discriminant validity. The concurrent validity was established by administering another psychometrically strong brief battery measuring cognitive impairements [Montreal Cognitive Assessment Battery (MoCA)] along with N-CABS on 16 patients. The findings of concurrent validity suggested that Cronbach alphas for 13 MoCA items and 8 N-CABS items came out to be 0.82  whereas the second one containing items related to visual skills . The division of subtests into two factors was ideal and in accordance with other renowned cognitive assessment batteries like Montreal Cognitive Assessment (MoCA),N-CABS has potential advantages over other neuropsychological screening tools like MMSE, MoCA or Cognistat. As it includes tasks which are more relevant to our culture, for example, in Naming subtest all animals are more common unlike animals used in MoCA. In the same way, these instruments require fine motor skills in some items (cube or clock drawing) limiting its use with individuals with motor deficits in stroke.The study favored the presence of PSCI among stroke survivors which were rigorously detected by N-CABS. The findings of the current study supported a significant prevalence of PSCI among stroke patients. The results elucidated that approximately 85% of the patients were detected to have PSCI. In Pakistan, very few studies exist determining PSCIs among stroke survivors. However, one of the studies aimed at assessing the prevalence of PSCI found that approximately 26% of the patients were having PSCI 26The basic purpose of this study was to evaluate a clinically and culturally sound neuropsychological test battery to assess cognitive impairments among stroke patients. Overall, N-CABS was found to be a reliable screening tool to assess six cognitive impairments including Attention, Memory, Naming, Arithmetic, Reasoning and Visual Neglect. It has several advantages over other bedside cognitive tests commonly used to detect PSCI as it is inexpensive, less time consuming and more culturally relevant. In future, a more detailed clinical diagnosis of etiology, volume and site of the lesions could be taken into account to control confounding in the results and get a clearer picture of the pattern of cognitive dysfunctioning. The present study was not able to develop extensive norms due to small sample size of patients, therefore, follow up studies should be planned on norm development of N-CABS. Lastly, the scope of current research was limited to cognitive functioning, future researches should expand the scope to emotional, psychological and motor dysfunctions.The current research has been done as a part of graduation project. Therefore, this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. There are no conflicts of interest to disclose.SNM conceived and designed study, did literature search, collected and analyzed data, did manuscript writing and editing.UB helped in conceptualization of study, statistical analysis and revision of initial and final manuscript.


We further demonstrated that the knockout efficiency could be greatly improved by using multiplex sgRNAs target the same gene. The F0 mutated animals showed fully loss-of-function by both genotyping and phenotyping analysis, which could enable direct functional analysis in the founders and avoid sophisticated breeding. This study not only presented the high efficiency of single base editing in a gigantic animal genome (>20 G), but also provided new tools for interrogating gene function in other salamander species.Loss-of-function approaches provide strong evidence for determining the role of particular genes. The prevalent CRISPR/Cas9 technique is widely used to disrupt target gene with uncontrolled non-homologous end joining after the double strand breaks, which results in mosaicism and multiple genotypes in the founders. In animal models with long generation time such as the salamanders, producing homozygous offspring mutants would be rather labor intensive and time consuming. Here we utilized the base editing technique to create the loss-of-function F0 mutants without the random indels. As a proof of principle, we successfully introduced premature stop codons into the  The salamanders comprise a group of more than 700 species of amphibians and constitute the order Caudata . The ordThe prevalent gene editing tool, clustered regularly interspaced short palindromic repeats-associated nucleases (CRISPR)/Cas, was widely used to disrupt gene function. It creates DNA double-strand breaks (DSBs) followed by non-homologous end joining (NHEJ) to generate random indels which leads to functional loss of the gene. Previous studies have applied CRISPR/Cas knockout to the axolotls and newts and yielded excellent insights into the underlying mechanisms responsible for the unique features of salamanders ,7,8, parPleurodeles waltl). The gene inactivation is realized by precisely converting the four codons  into stop codons , by the cytidine deaminase (C-to-T) in the base editor complex, and subsequently the early termination of the translation. We initially focused on the single copy newt tyrosinase (tyr) gene as its protein product is the rate-limiting enzyme in the synthesis of melanin [tyr locus could produce albino animals with no pigmentation and develop normally, which is a convenient phenotypic readout of successful gene disruption. We designed sgRNAs to target the first exon of tyr gene and utilized both the canonical CRISPR/Cas9 (indel mutation) and the latest base editing means (iSTOP) to achieve loss-of-function. Our results showed that the base editing technique and multiplex sgRNA strategy could efficiently induce iSTOP to inactivate the tyr gene to create albinism phenotype. The base editing is a potent way to safely and stably generate gene modified salamanders. In this study, we tested the possibility of base editing in salamanders by using the induction of stop codons (iSTOP) knockout strategy in the Iberian ribbed newt  was employed to synthesize the sgRNAs. The IVT was performed according to the manufacturer’s instructions. The sgRNAs concentration was adjusted to 50–100 ng/μL and sgRNAs was kept at −80 °C until use. The fourth generation of base editor was used in this study. pCMV-AncBE4max (AncBE4max) was a gift from Dr. David Liu (Addgene plasmid # 112094). The base editing window of AncBE4max was around positions four to eight of the protospacer, counting the PAM site as positions 21–23. The plasmid was linearized by using restriction enzyme BbsI and purified with ethanol precipitation. In vitro transcription (IVT) was performed with mMESSAGE mMACHINE T7 ULTRA kit . The transcribed mRNA was purified by lithium chloride precipitation and the concentration was adjusted with RNase-free water. All the sgRNAs are targeting Pleurodeles waltl) were kept in tap-water tanks at 22–24 °C under natural light cycles. The larvae were fed with hatched artemia every day and the juvenile/adult newts were fed with frozen blood worms and pellets every other day. Natural breeding was promoted and the egg laying was induced by human chorionic gonadotropin injection of females [Iberian ribbed newts  gene (tyr locus by microinjecting the SpCas9 mRNA and sgRNAs into fertilized newt eggs (single-stage embryos). The CRISPR/Cas9 system can continuously operate at the target during embryonic development, leading to mosaicism of the introduced mutations. If the CRISPR/Cas9 destroy the tyr gene, the pigmentation phenotype can be visually identified. Thirteen days later, we observed albino animals in three out of five groups of sgRNA injected embryos, indicating that only the sgRNA#3, #4, and #5 are operative. The knockout efficiency varies in different groups . Then we extracted DNA from individual animals from each group and PCR amplified the target region. Indeed, the sanger sequencing chromatograms showed complicated mosaic mutations adjacent to the PAM site, indicate multiple indels occurs in the same animal (The detailed scheme of the iSTOP design was shown in yr) gene A. We fire animal B. AccordTo explore whether the base editing tool is working in newt, we performed similar procedures by co-injecting AncBE4 mRNA and sgRNAs into fertilized eggs. After PCR sequencing, we identified C-to-T conversions in the specific loci as expected in four out of five  groups A. HoweveNext, we examined the pigmentation in the eyes as the reference to determine the knockout efficiency in the base edited animals. A total of 10%, 66%, and 13% of animals injected with sgRNA#3, #4, and #5 showed missing melanin in at least one eye . In compAfter verifying the actions of sgRNAs and AncBE4max on iSTOP, we continued to test whether the multiple sgRNAs strategy is possible to improve the efficiency of gene deletion. The selected sgRNA#3, #4, and #5 were injected into the one-cell embryos separately or combined while the AncBE4max mRNA amount remain constant. At 13-day post injection, we found that the single sgRNAs can induce at most half of animals to completely lose the pigmentation, but multiple sgRNAs together can produce 70% of the animals with fully loss of pigment. Co-injection of multiple-sgRNAs with “high concentration” has similar gene disruption effect compare to “low concentration”, these results indicate that the improving of knockout efficiency is not due to the increase of the substance of sgRNAs, but to multiple-sgRNAs (multiple stops) strategy . The DNAAs detailed above, the base editing works efficiently in newts by inducing targeted C-to-T conversion that can introduce a premature stop codon into a protein-coding sequence, providing a means of generating loss-of-function mutants. Current method presents several advantages compared to the canonical CRISPR/Cas9 mediated gene disruption technology. First, base editing does not elicit any DSBs, whose formation is particularly deleterious in the early stage of animal development . Furthertyr-base edited animals developed and metamorphosed normally compare to the wild type ones (Latest studies showed that cytosine but not adenine base editors have undesired off-target effects on DNA in mice  and riceype ones , indicattyr base editing could serve as the technical control. To our knowledge, this report presents the evidence showing the largest animal genome ever been successfully base edited. With the constant engineering to the base editors to improve the editing window, efficiency, and accuracy, the next generations of base editing technique will undoubtedly be safer and easier to use . The curAltogether, our work established the base editing mediated iSTOP as a robust and efficient gene disruption technology in newt and provided invaluable tools to analyze the interesting genes identified from the growing omics data of different salamander studies.


Clinical application of rivaroxaban and apixaban does not require therapeutic monitoring. Commercial anti-activated factor X (anti-FXa) inhibition methods for all anti-FXa drugs are based on the same principle, so there are attempts to evaluate potential clinical application of heparin-calibrated anti-FXa assay as an alternative method for direct FXa inhibitors. We aimed to evaluate relationship between anti-FXa methods calibrated with low molecular weight heparin (LMWH) and with drug specific calibrators, and to determine whether commercial LMWH anti-FXa assay can be used to exclude the presence of clinically relevant concentrations of rivaroxaban and apixaban.Low molecular weight heparin calibrated reagent  was used for anti-FXa activity measurement. Innovance heparin  calibrated with rivaroxaban and apixaban calibrators  was used for quantitative determination of FXa inhibitors.Analysis showed good agreement between LMWH calibrated and rivaroxaban calibrated activity (κ = 0.76) and very good agreement with apixaban calibrated anti-Xa activity (κ = 0.82), respectively. Low molecular weight heparin anti-FXa activity cut-off values of 0.05 IU/mL and 0.1 IU/mL are suitable for excluding the presence of clinically relevant concentrations (< 30 ng/mL) of rivaroxaban and apixaban, respectively. Concentrations above 300 ng/mL exceeded upper measurement range for LMWH anti-FXa assay and cannot be determined by this method.Low molecular weight heparin anti-FXa assay can be used in emergency clinical conditions for ruling out the presence of clinically relevant concentrations of rivaroxaban and apixaban. However, use of LMWH anti-FXa assay is not appropriate for their quantitative determination as an interchangeable method. Direct oral anticoagulants (DOACs) have been increasingly used for the prevention and treatment of thromboembolic diseases in recent years. Compared to vitamin K antagonists (VKAs), clinical application of DOACs does not require routine coagulation monitoring. However, according to the present expert opinions, there are special clinical situations in which laboratory measurement of DOACs in plasma should be performed, including bleeding or thromboembolic events (acute stroke), emergency surgical or invasive procedures, extremes of body weight, renal and/or liver failure resulting with reduced drug elimination and suspected non-compliance or overdose  and activated partial thromboplastin time (APTT). Knowing the impact of DOACs on the results of screening coagulation tests is a precondition for the correct interpretation of these assays. However, PT and APTT are not appropriate for direct FXa inhibitors, neither for quantifying drug concentration and reliable assessment of their anticoagulant effect, nor to exclude the presence of clinically relevant drug concentrations in the circulation due to the high differences in sensitivities of individual commercial PT and APTT reagents .Samples from patients taking rivaroxaban and apixaban were collected from July to December 2018 at the Department of Neurology and Department of Cardiovascular Diseases, Sestre Milosrdnice University Hospital Center. A total of 61 samples from patients taking rivaroxaban (31 peak and 30 trough) and a total of 53 (30 peak and 23 trough) samples from patients taking apixaban were used in the study. Blood samples were taken from the same patients and on the same day to obtain both, trough (immediately prior the next drug dose) and peak (two hours after drug administration) concentrations of rivaroxaban and apixaban in plasma. All patients were treated with standard and equal drug doses for non-valvular atrial fibrillation (NVAF) clinical indication  containing 3.2%-trisodium citrate (volume 3.5 mL). All samples were centrifuged at room temperature for 10 minutes at 1800xg to obtain platelet poor plasma, aliquoted into labelled tubes and stored at - 20°C until analysis. Samples for the conducted study have been chosen in order to cover as much as possible the whole measurement range (up to 500 ng/mL) for both rivaroxaban and apixaban concentrations.All coagulation assays were performed on Behring Coagulation System XP (BCSXP) analyser . Low molecular weight heparin anti-FXa activity was determined in all samples by chromogenic method using original manufacturers´ reagent kit  and LMWH calibrator . Results were expressed in anti-FXa heparin equivalent international units (IU/mL). The concentrations of rivaroxaban and apixaban were measured using specific chromogenic anti-FXa assay , calibrated with specific calibrators for rivaroxaban and apixaban . Concentrations of rivaroxaban and apixaban were expressed in ng/mL.et al.  analysis was done to determine cut-off values of LMWH calibrated anti-FXa activity which corresponds to rivaroxaban and apixaban values < 30 ng/mL and < 50 ng/mL. Those cut-off values were used as suggested for the treatment of patients with excessive bleeding and perioperative management by Levy The rivaroxaban concentrations obtained by chromogenic anti-FXa method with drug specific calibrators ranged from 62 to 433 ng/mL for peak and from 4 to 83 ng/mL for trough concentrations. The apixaban peak and trough concentrations ranged from 73 to 415 and from 13 to 98 ng/mL, respectively.Results of rivaroxaban and apixaban peak and trough concentrations as well as LMWH anti-FXa activites are presented in Kappa statistics showed good agreement between LMWH calibrated and rivaroxaban calibrated FXa activities (κ = 0.76) . FurtherAs presented in In this study, we evaluated relationship between two chromogenic anti-FXa methods, one calibrated with LMWH and the other with drug specific calibrators for rivaroxaban and apixaban. The main two questions that we wanted to answer were whether LMWH-calibrated anti-FXa activity assay can be used: 1) for excluding clinically relevant concentrations of direct anti-FXa drugs in circulation and 2) for quantifying rivaroxaban or apixaban concentrations in plasma as an alternative method to the chromogenic assays calibrated with specific drug.et al. who reported that anti-FXa response is not the same at the concentrations of 30 and 50 ng/mL of rivaroxaban and apixaban  of the International Society on Thrombosis and Hemostasis (ISTH) recommended administration of the reversal agent in the perioperative setting, if plasma concentration of direct FXa inhibitor is above 30 ng/mL, in order to ensure adequate haemostasis , whereas recommended measurement unit for direct FXa inhibitors is ng/mL. Further, there is an evidence of substantial variability between different commercial kits and calibrators for LMWH-calibrated anti-FXa assays  used in anti-FXa assays intended for different anti-FXa drugs, a group of authors have recently proposed a new concept that could contribute in solving this problem. These authors suggested a concept for a single anti-FXa based laboratory assay for all drugs that directly or indirectly inhibit FXa. The initiators of the idea of so called „Da-Xa inhibition assay“ suggest a new test that would report inhibitory activity rather than drug concentration or IU/mL (in vitro. Those results are significantly different from the values in the plasma of patients treated with DOACs (The strength of this study relies on the fact that the results are obtained in plasma of patients treated with both direct anti-FXa inhibitors, rivaroxaban and apixaban, unlike several previous studies that reported results of drug concentrations in plasma samples where the certain concentration of DOACs was added The limitation of our study is that it was restricted to NVAF as the only clinical indication included. However, this clinical condition still represents the most common indication for introduction of DOACs in patient management, thus being the real representative patient population for the purpose of this research. Furthermore, a possible limitation could be the fact that we compared only one LMWH-anti-FXa assay with one drug-calibrated anti-FXa assay for rivaroxaban and apixaban available in the market. However, our intention was to compare the two methods that are in use at our laboratory in order to apply results in management of patients treated at our institution.In conclusion, the findings of this study will improve the understanding in terms of both possibilities and limitations of LMWH-calibrated chromogenic anti-FXa assays in patients treated with direct anti-FXa inhibitors. Our results strongly suggest that LMWH calibrated anti-FXa assay has a potential as an alternative first line method for excluding the presence of significant levels of rivaroxaban and apixaban if laboratory has no available specific chromogenic anti-FXa assay calibrated with particular drug. However, in case of positive result suggesting the presence of an oral anti-FXa inhibitor in plasma, specific chromogenic assay for particular anti-FXa drug should be performed for quantitative determination of these drugs. The use of LMWH calibrated anti-FXa assay to quantify rivaroxaban and apixaban concentrations could not be recommended in routine clinical practice as the only method for quantification of anti-FXa medications. It is of crucial importance that both laboratory experts and clinicians who treat patients substantially understand the opportunities and limitations of heparin-calibrated anti-FXa assays in patients treated with anti-FXa inhibitors.


Drosophila melanogaster, we utilized a set of transgenic Gal4-dependent competitive inhibitors  to functionally assess ca. 140 high-confidence Drosophila microRNAs using automated quantitative movement tracking systems followed by multiparametric analysis. Using ubiquitous expression of miR-SP constructs, we identified a large number of microRNAs that modulate aspects of normal baseline adult locomotion. Addition of temperature-dependent Gal80 to identify microRNAs that act during adulthood revealed that the majority of these microRNAs play developmental roles. Comparison of ubiquitous and neural-specific miR-SP expression suggests that most of these microRNAs function within the nervous system. Parallel analyses of spontaneous locomotion in adults and in larvae also reveal that very few of the microRNAs required in the adult overlap with those that control the behavior of larval motor circuits. These screens suggest that a rich regulatory landscape underlies the formation and function of motor circuits and that many of these mechanisms are stage and/or parameter-specific.Locomotion is an ancient and fundamental output of the nervous system required for animals to perform many other complex behaviors. Although the formation of motor circuits is known to be under developmental control of transcriptional mechanisms that define the fates and connectivity of the many neurons, glia and muscle constituents of these circuits, relatively little is known about the role of post-transcriptional regulation of locomotor behavior. MicroRNAs have emerged as a potentially rich source of modulators for neural development and function. In order to define the microRNAs required for normal locomotion in  Caenorhabditis elegans and Drosophila melanogaster have been applied to the assembly of these motor circuits  have emerged as a rich potential source for modulation of nervous system function . These sDrosophila suggest that multiple aspects of synapse architecture, remodeling and/or function are under post-transcriptional regulation  development in C. elegans in which miRNAs and miRNA families were mutated has suggested that a relatively small number of miRNAs are essential for locomotion in this organism  transgenic lines designed to inhibit the function of ca. 140 high confidence miRNAs . The effubiquitous expression, and then to explore the underlying temporal and spatial logic of their function using more selective expression. Since the adult and larval stages are fundamentally different in body plan and locomotor strategy, we screened both second instar larvae and mature adults. For miR-SP lines that gave adult phenotypes, we used tsGal80 to determine which miRNAs were acting to regulate development of adult circuits and which were required for ongoing adult function. These screens yielded a surprisingly large number of miRNAs that modulate different aspects of spontaneous baseline locomotion. Most appeared to function within the nervous system and to affect developmental processes. The majority of miRNAs that had exclusively adult function displayed phenotypes which suggested involvement of the miRNA in specific locomotor parameters as opposed to global regulation of behavior. Our analysis provides important new information on the complexity of post-transcriptional regulation in the development and function of motor circuits. Understanding the role of miRNAs in locomotor circuits will also be critical for the interpretation of miRNA phenotypes in other behaviors that may have motor outputs. Thus, we present this data as a resource for others who hope to use behavioral approaches to map miRNA functions in the future.Our aim for the current study was to capture the broadest set of potential regulators using tubulin-Gal4 , C155-Gal4 (miR-SPs a mutation in Dicer-1 (1D14Dcr-1) was recombined onto the elav-Gal4 chromosome to generate an elav-Gal4, Dcr-11D14/balancer line. This line was crossed to homozygous UAS-miR-SP lines and mCherry-positive progeny used for assays. The use of a heterozygous Dcr-1 background weakens the RISC system, enhancing the effects of miR knockdown, a phenomenon we noted while carrying out the original screens  within the same 2-h window each day. Using mouth aspiration, single male flies were loaded into (40 × 10 mm) petri dish cover arenas lined and secured with Parafilm. Each chamber contained one fly. This allowed for a light-transmissive enclosure that was isolated from other external cues. Flies were allowed to freely explore their arenas for 5 min while being observed and recorded by video camera and tracking software at a 10 Hz resolution – see . Lines that were not significantly different from Scramble were excluded from further analysis. We then took the significantly different lines and analyzed them further using a Dunnet’s Test for each independent variable to find out where the deficiency was for each miR-SP compared to control. For continued inclusion, a line had to be significantly different from Scramble-SP for at least one locomotor variable. This analysis was necessarily conservative, as the initial MANOVA identified a large cohort of lines that were significantly different due to the combined effect of multiple parameters, yet failed to meet significance under a single univariate test. Thus, we have high confidence in phenotypes for the remaining lines. Finally, lines were grouped according to the type of locomotor deficiency: (1) those dealing with how active a fly was , or (2) those characteristics that relate to how far a fly traveled . The phenotypes induced by miR-SP expression were sorted into the “active”, “distance”, or “both” categories (see Discussion).The data files were processed and analyzed using custom-made software as described previously  over a grape juice-supplemented agar 60 × 15 mm plate, and a droplet of re-hydrated yeast for 2-4 h at 25° and 80–90% relative humidity. Collection plates with fertilized eggs were incubated for 72-80 h under the same conditions. 2nd instar larvae, identified by anterior and posterior spiracle and mouth hook morphology, were gently washed with 1X phosphate saline buffer and placed onto dry agar plates for mCherry expression screening; GenII miR-SPs were designed with mCherry as a reporter of SP cassette expression . The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures, and supplemental tables. Statistical data for behavioral assays was uploaded to GSA figshare portal. Supplemental material available at figshare: Drosophila  individually for many miRNA genes. Having recently demonstrated the efficacy of competitive inhibition using miR-SP technology , we emplBecause miRNAs often function to tune the expression of downstream genes within narrow limits , we requCanton-S and 1118w) and the Gal4 driver strains to assess the effects of genetic background. Our initial screen was designed to examine ubiquitous inhibition of miRNA activity using the tub-Gal4 driver. We detected a subtle yet consistent background effect of the tub-Gal4 driver compared to Canton-S wild type. However, the tub-Gal4;Scramble-SP control was not significantly different from tub-Gal4 across multiple locomotor parameters. This highlights the importance of genetic background and indicated that the most informative “wild type” control genotype for our screen was tub-Gal4;Scramble-SP rather than the more standard Canton-S or 1118w.Before initiating a genetic screen of miR-SP strains, we compared baseline locomotion in the adult assay with standard wild type control strains  using Multivariate Analysis of Variance (MANOVA) to identify lines that displayed abnormal locomotor behavior compared to tub-Gal4;Scramble-SP controls. Because some miR-SP lines display penetrant lethal phenotypes when crossed to tub-Gal4 , whereas three lines were specific to distance . The largest category of parameter-specific phenotypes was average acceleration, which includes nine miR-SP lines  strain and grown at permissive temperature (18°) until eclosion, followed by a shift to 29° to allow tub-Gal4 to be active for a period of several days prior to behavior assay. Of the 137 miR-SP lines tested with this regimen, only three miR-SPs displayed significant effects on one or more of six quantitative parameters: miR-263bSP, miR-282SP, and miR-994SP. Each of the three phenotypes was distinct from the phenotype obtained with tub-Gal4 . We assembled a video-tracking arena illuminated with a wavelength invisible to larvae in order to record spontaneous locomotor behavior  of our miR-SP lines induced a significant effect on one or more locomotor parameters in L2 stage larva . This peDrosophila, we utilized conditional competitive inhibition and unbiased quantitative behavior tracking to test the function of ca. 140 miRNAs at two developmental stages. Our data suggest that a surprisingly large number of miRNAs modulate locomotion in adults and in larvae. However, comparisons between miRNAs required for normal locomotion at different developmental stages demonstrate that few miR-SPs induce phenotypes in both larvae and adults  have uncovered conserved signal transduction pathways and transcriptional programs that guide development and tune the ongoing activity of the circuit (in vivo miRNA screens for synapse development have yet been described, recent studies in Drosophila have identified several conserved miRNAs that regulate the development, form and/or function of neuromuscular synapses (miR-124SP even though independent miR-124 nulls display locomotor defects (In addition to bringing many insights into developmental mechanisms ( circuit . Althougsynapses . NotablyDrosophila larva (Our finding that some miRNAs control distinct aspects of locomotor behavior raises the question of whether these miRNAs may be under dynamic control as a means to tune behavioral state, or whether these miRNAs specify a static baseline state of the motor circuit. Recent expression profiling identified a number of activity-responsive miRNAs in la larva . Such miIt is also possible that certain activity-dependent miRNA mechanisms may be essential for the refinement of locomotor patterns. Optimal performance of motor systems appears to involve an initial experience-dependent phase at both the larval and adult stage (Drosophila will offer the ability to detect miRNA roles in regulating a range of behaviors (tub-Gal4 screen, including miR-9a, miR-11, miR-210, miR-276b and miR-282. This is a large overlap in light of the fact that our multi-parametric data analysis was designed to detect subtle modulatory effects on distinct aspects of movement. A subsequent screen of 81 of the miRNA deletions for larval self-righting behavior in first instar larvae revealed that over 40% of these chronic and systemic mutants display defects (The advent of genetic tools for comprehensive loss-of-function screens in It is tempting to speculate that the large number of miRNA genes revealed by these screens may reflect distinct regulatory activities in the different classes of neurons and/or glia that contribute to the motor circuits of larva and adults. As dissection of the motor circuitry advances and Gal4 drivers become available to test each constituent cell type (


Oncorhynchus mykiss) that also differ in lifetime fitness. Developmental plasticity, which can also affect fitness, may be mediated by epigenetic mechanisms such as DNA methylation. Our previous study identified significant differences in DNA methylation between adult hatchery- and natural-origin steelhead from the same population that could not be distinguished by DNA sequence variation. In the current study, we tested whether hatchery-rearing conditions can influence patterns of DNA methylation in steelhead with known genetic backgrounds, and assessed the stability of these changes over time. Eyed-embryos from 22 families of Methow River steelhead were split across traditional hatchery tanks or a simulated stream-rearing environment for 8 months, followed by a second year in a common hatchery tank environment. Family assignments were made using a genetic parentage analysis to account for relatedness among individuals. DNA methylation patterns were examined in the liver, a relatively homogeneous organ that regulates metabolic processes and somatic growth, of juveniles at two time points: after eight months of rearing in either a tank or stream environment and after a subsequent year of rearing in a common tank environment. Further, we analyzed DNA methylation in the sperm of mature 2-year-old males from the earlier described treatments to assess the potential of environmentally-induced changes to be passed to offspring. Hepatic DNA methylation changes in response to hatchery versus stream-rearing in yearling fish were substantial, but few persisted after a second year in the tank environment. However, the early rearing environment appeared to affect how fish responded to developmental and environmental signals during the second year since novel DNA methylation differences were identified in the livers of hatchery versus stream-reared fish after a year of common tank rearing. Furthermore, we found profound differences in DNA methylation due to age, irrespective of rearing treatment. This could be due to smoltification associated changes in liver physiology after the second year of rearing. Although few rearing-treatment effects were observed in the sperm methylome, strong family effects were observed. These data suggest limited potential for intergenerational changes, but highlight the importance of understanding the effects of kinship among studied individuals in order to properly analyze and interpret DNA methylation data in natural populations. Our work is the first to study family effects and temporal dynamics of DNA methylation patterns in response to hatchery-rearing.Genetic selection is often implicated as the underlying cause of heritable phenotypic differences between hatchery and wild populations of steelhead trout ( Oncorhynchus mykiss), with fish reared at 3 °C above normal temperature returning two weeks later on average than those reared at a normal temperature [In fish, traits such as growth, sex differentiation, life history and behavioral performances can be influenced by the environmental conditions encountered during development . Many ofperature . While operature ,15,16.cyp19a) promoter and subsequent predominance of the male phenotype [myogenin, a gene with important functions in muscle development [Epigenetics refers to mechanisms that alter gene activity without modifications to the underlying DNA sequence . Epigenehenotype . In rainhenotype ,23. In Aelopment . Early lelopment .Oncorhynchus kisutch) from two independent rivers shortly after release from the hatchery. While genetic differentiation between hatchery- and natural-origin fish within a river was nonsignificant, substantial differences in DNA methylation in the muscle of juvenile hatchery and natural-origin coho were observed [The ability of early environmental information to be “stored” in the form of an epigenetic memory has also been explored recently in hatchery-reared salmonids. Two recent studies examined the potential for DNA methylation to store epigenetic information acquired in the hatchery-rearing environment as a first step toward understanding how epigenetic mechanisms may be contributing to loss of fitness phenotypes observed in hatchery-reared salmonids. Le Luyer et al.  examinedobserved . These robserved ,28,29 anobserved  addresseobserved , in thatobserved . While bobserved .Immediate hereafter), (2) Are differences in DNA methylation as a result of hatchery rearing persistent after 12 months of rearing in a common environment? (referred to as Persistent hereafter), (3) Are there developmental changes in DNA methylation in the same tissue between age-1 and age-2? , (4) What is the potential for intergenerational transmission of DNA methylation changes as a result of hatchery rearing? . Addressing this set of questions allowed us, for the first time, to examine the dynamics of hatchery-induced changes in DNA methylation across multiple life-stages and tissues in steelhead; addressing a significant gap in previous studies.Here we used a controlled experiment to investigate whether hatchery-rearing conditions can influence patterns of DNA methylation in steelhead and directly assessed the stability of these changes over time in fish with known genetic backgrounds. For this experiment, embryos from 22 families of Methow River steelhead were split across two rearing treatments; either traditional hatchery rearing or a simulated stream rearing environment. The families were reared for 8 months before merging the two treatments for an additional 12 months of rearing in a common tank environment. DNA methylation patterns were examined in the liver of male juveniles at the end of the rearing treatment (age-1) as well as in sexually immature males after common hatchery rearing (age-2). Further, we analyzed DNA methylation in the sperm of males that matured at age-2 to assess the potential of environmentally-induced changes to be passed to offspring. With this experimental design, we were able to specifically investigate four questions: (1) Are there immediate changes in DNA methylation after eight months of hatchery rearing?  were analyzed to test for developmental changes in DNA methylation  had two natural-origin parents. Gametes from mature adult steelhead were manually collected and eggs were fertilized using typical hatchery protocols between 15 April and 5 May 2014. Developing embryos were incubated in Heath trays at the Winthrop National Fish Hatchery . Incubation temperatures were manipulated to synchronize emergence timing among progeny from different spawning dates to minimize effects of emergence timing on feeding and growth performance after exogenous feeding. Specifically, eggs were held on ambient well water (~7.8 °C) for approximately 2, 7 or 21 days before being slowly (hours) transitioned to chilled (~3.9 °C) water depending on fertilization date . See A total of 21 female  and 20 male  adult steelhead returning to the upper Methow River  were used to create 22 families used in this study. Methow River summer-run steelhead are part of an Upper Columbia River evolutionary significant unit (ESU) of West Coast steelhead populations currently listed as threatened under the Endangered Species Act. A majority of the families were single-pair matings, but there was also a single pair of paternal half-sib families and a single pair of maternal half-sibling families. All families had at least one natural-origin parent and a majority of the families  where each family was loaded into a 4-inch diameter PVC isolette. The isolettes were randomly placed into Heath trays. The single stack of Heath trays received approximately 12 L per minute of well water (10.4 °C). The Heath stack was wrapped with black plastic to eliminate most ambient light. The embryos were checked the following day and there were no mortalities.On 11 June 2014, 40 eggs from each family were divided equally among four containers . Groups of 210 eggs from each of the four containers were deposited into four artificially constructed nests in a 40 m-long by 6 m-wide outdoor simulated natural stream channel (described in ). The chOn 8 August 2014, juvenile steelhead at the WNFH had reached the “button-up” stage of development; the embryos had utilized all available yolk resources during rearing in typical hatchery stack incubators. At this time, we transferred an additional 20 fry from each of the same families previously stocked in the stream channel at Manchester. The fry were transported to the Manchester Research Station in two insulated coolers, with each cooler containing 10 fry from each female. Each cooler of fry was stocked into a separate circular fiberglass tank (1.8 m diameter × 0.6 m deep) plumbed into a recirculating freshwater system with an annual average rearing temperature of 9.4 °C. Average daily temperatures ranged between 11.6 °C (August) and 7.6 °C (February) over the rearing period . Fry were fed daily to excess for the first three weeks of culture and then fed to satiation five days per week for the remainder of the culture period in order to produce the highest growth rates and highest rates of precocious male maturation possible. Juvenile steelhead in the hatchery-rearing treatment included fish that were also part of another study that required periodic handling for size measurements and tagging. Fish were anesthetized in buffered 0.01% tricaine methanesulfonate  prior to size measurements in October, November and December 2014, and February and May 2015.n = 117 total ) were too small to PIT tag in February 2015 were subsequently PIT tagged in May 2015 and included in subsequent analyses. A two-tailed t-test was used to detect differences in size between stream- and hatchery-reared individuals.In February 2015, a total of 624 fish, 237 stream-reared and 387 hatchery-reared, were weighed (to nearest 0.1 g), measured (fork length (FL) to nearest mm) and implanted with 9 mm PIT tags. Fin clips were taken for DNA isolation to determine genetic sex and family. A subset of the smallest stream-reared individuals (n = 10) and stream-reared (n = 10) fish were used for RRBS.To address question 1, we examined DNA methylation in liver tissue fish at 10 mpf (referred to hereafter as “age-1” fish). Liver tissue was selected because it is a relatively homogeneous tissue and plays a central role in the endocrine control of somatic growth as well as all metabolic processes controlling storage and mobilization of nutrients. These physiological characteristics are likely to differ between fish reared in hatchery and natural environments because of differences in diet, water chemistry and temperature. Prior to transfer of fish from stream channel to tanks, a subset of 20 fish per treatment were lethally subsampled to collect liver tissue for reduced representation bisulfite sequencing (RRBS) analysis. Fish were anesthetized in 0.01 % MS222 and euthanized by decapitation. Parentage was unknown at the time of liver sampling; fish selected for lethal sampling were size matched as much as possible. Gender was determined by gross morphology of the gonad and genetic sex was determined by analysis for the male sex determining gene . A portn = 10 each from the hatchery- and stream-rearing treatments) for RRBS analysis in order to assess the persistence of DNA methylation changes in liver tissue resulting from hatchery rearing. A portion of posterior lobe of the liver was dissected and flash frozen in liquid nitrogen until DNA isolation.After subsampling in February 2015, all remaining fish were placed into a common environment (hatchery tanks) for an additional year to test for the persistent effects of hatchery-rearing on DNA methylation in liver of immature males and sperm of males that matured by 20 months of age (referred to hereafter as “age-2” fish). Rearing of fish in the simulated stream environment was limited to eight months because natural productivity was not sufficient to support the biomass of fish as they continued to grow. Hatchery-reared fish were distributed into two tanks  while stream-reared fish distributed into 3 tanks to separate the smallest individuals. The stream-reared fish were then transitioned to a size appropriate pelleted commercial trout diet. Pelleted feed had the same nutritional composition regardless of pellet size. Both the hatchery and stream-reared fish were fed to satiation 5 days a week in order to promote precocious maturation of males in both experimental treatments. In September of 2015, all fish were weighed and measured. A majority of the females (identified by genetic sex test described below) were removed from the study to decrease density and males were distributed back into the tanks. In February of 2016, a subset of immature males was lethally sampled  loci, following the methods described by the authors of . The resGenomic DNA was isolated from male livers using the DNeasy Blood and Tissue Kit  following manufacturer’s instructions. DNA was ethanol precipitated prior to quantification using the Quant-iT PicoGreen dsDNA Assay Kit . A subset of representative DNA samples was analyzed to check DNA quality on the Agilent 2200 TapeStation .Milt was expressed by gentle abdominal pressure, collected with capillary action using Natelson tubes, transferred to microfuge tubes and frozen in liquid nitrogen. Genomic DNA was isolated from diluted milt (2 uL milt in 598 uL PBS) using the DNeasy Blood and Tissue Kit (Qiagen) following a user-defined protocol for DNA isolation from sperm. DNA was quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen). A subset of representative DNA samples was analyzed for quality on the Agilent 2200 TapeStation .n = 7–19 samples per pool). Two rounds of bisulfite conversion were performed using the EpiTect Bisulfite Kit (Qiagen), followed by PCR amplification (TruSeq DNA LT Library Prep Kit (Illumina)) and final clean-up using Agencourt AMPure XP beads (Beckman Coulter). Pooled libraries were sequenced for 100 cycles (single-end reads) in 2–4 lanes  on an Illumina HiSeq. The Immediate and Intergenerational samples were sequenced on a HiSeq 2500 ) and Intergenerational libraries were sequenced both Omega Bioservices and the University of Oregon, Genomics and Cell Characterization Core Facility . Persistent libraries were sequenced on a HiSeq 4000 at the University of Oregon, Genomics and Cell Characterization Core Facility. Sequencing data are available in the NCBI SRA database under the BioProject PRJNA525072.DNA was prepared for RRBS using the gel-free technique as described in . BrieflyTrimGalore  a wrapper for the publicly available trimming tool cutadapt [--rrbs. Trimmed reads were aligned to the O. mykiss reference genome scaffolds . Count data for methylated and unmethylated reads were extracted using the Bismark methylation extractor script for downstream analysis. In order to screen for batch effects between sequencing platforms and facilities, bisulfite treatment efficiency was estimated using non-CG methylation and a two-tailed t-test was performed. Furthermore, all libraries were evaluated with FastQC and reports visualized using multiQC [Sequencing reads were quality and adapter trimmed using cutadapt  and Fastcutadapt . TrimGal163495.1 ) with th163495.1 . Bismark multiQC  to identImmediate (n = 10 each per rearing group), Persistent (n = 10 each per rearing group), Developmental (n = 20 each per time-point), Intergenerational (n = 30 each per rearing group). Filtering steps and differential methylation analyses, described in detail below, were performed for each contrast separately in order to retain the highest number of CG sites for differential methylation analyses.Differential methylation analyses were performed for each of the four questions/sample contrasts: StAMMP [Immediate, Persistent and Intergenerational sample contrasts. For the Developmental contrast, time-point (age-1 or age-2) was used as the predictor variable and rearing-environment was used as a model covariate to account for differences that may be specific to a given rearing environment. Multiple hypothesis testing was performed on P-values extracted from the MACAU output for each CG site using the false discovery rate (FDR) approach used in the R package qvalue [Immediate and Persistent sample contrast. Because CG sites were filtered separately for each contrast, we evaluated the amount of overlap between CGs analyzed between these sample contrasts. The genomic location of all CG sites analyzed as well as DMCs for the Immediate and Persistent time-points were compared for overlap using the R package dplyr [For each contrast, CG sites that were covered in at least half of the samples per rearing group were considered for analysis. In addition, CG sites with median coverage ≤10, CG sites that had ≥90% or ≤10% average DNA methylation across all samples or were in the lowest 5% of variance in methylation among samples were filtered out prior to modeling differential methylation. Due to the inability of bisulfite sequencing technologies to discern a true C/T SNP from a methylation change, a final filtering step was performed to remove sites with a putative CG SNP. BS-SNPer  was usedStAMMP  based one qvalue . We conse qvalue . We alsoge dplyr .P-value of ≤0.001, or, (2) three or more CG sites with a P-value < 0.001 within a 2 kb region.We also identified differentially methylated regions (DMRs)—spatially co-located stretches of differentially methylated CGs, which are more likely to regulate gene expression than differentially methylated sites occurring in isolation . MeetingO. mykiss reference genome (NCBI assembly accession: GCF_002163495.1). DMCs and DMRs overlapping genes and their putative cis regulatory domains (defined here as within 10 kb of the transcription start site or transcription end site) were identified using the software BEDTools [http://uniprot.org) in order to determine homology to known protein sequences. Alignments were made using the BLAST algorithm [−10 e-value cutoff). Pathway and network analysis were conducted on genes associated with DMCs using Qiagen’s Ingenuity Pathway Analysis (IPA). To analyze in IPA, genes were assigned orthologs using the ENSEMBL zebrafish (GRCz10 release 84), ENSEMBL human (GRCh38 release 84), and Swiss-Prot databases using BLAST (v2.6.0+). Orthologs were then checked with IPA’s internal database to find a match. If multiple databases had a match, the order of selection was zebrafish, human then Swiss-Prot. A gene was only assigned an ortholog if IPA found a match with the ortholog’s accession number and the BLAST e-value was ≤ 1 × 10−5. All genes within 10 kb of an analyzed CG site were used as the background for enrichment analysis. Significantly altered pathways were defined as those with a Fisher’s Exact Test  with a minimum number of hits per pathway cutoff of 3.All DMCs and DMRs were annotated to genes using predicted gene models for the BEDTools ,51). Genlgorithm  , but stream-reared males generally tended to be shorter than hatchery-reared males and also showed more variation  separately. Intergenerational sample set used DNA from sperm where a large proportion of the CG sites had average methylation >90% across all samples. These CG sites were filtered out prior to differential methylation analysis in MACAU, and as a result, only a quarter of the number of CG sites were interrogated for this sample set. Bisulfite sequencing is the gold standard of DNA methylation analysis, but it cannot differentiate between C/T SNPs. Here, approximately 20,000 CG sites were identified as possible C/T SNPs across the sample sets. These CG sites were removed from further analysis.In order to retain as many CG sites for analysis as possible, filtering criteria were assessed on each of the 4 sample contrasts  set were not significantly different between rearing groups. Eleven families were represented within this sample set; 6 of the families were represented in both hatchery and stream treatments. See Immediate time-point.Body size (weight and FL) of fish used in the n = 251) were hypo-methylated in hatchery-reared fish compared to stream-reared fish. Individuals clustered according to rearing-environment based on the 413 DMC  between hatchery- and stream-reared fish. The average absolute difference in methylation for DMCs was 25.7% (range: 8.3–65.5%). More than half of the DMC ( 413 DMC . Almost  413 DMC . Gene on 413 DMC . We iden 413 DMC .n = 9) and stream-reared (n = 10) male fish at the Persistent time-point. Ten families were represented within this sample set; 4 of the families were represented in both the hatchery and stream rearing groups. See Persistent time-point does not distinguish hatchery and stream-reared fish, in contrast to the sample set from the Immediate time-point  and Persistent (age-2) sample sets by analyzing DMCs using treatment as a covariate.Given the limited evidence to suggest a persistent signal of hatchery rearing in the liver after a year of rearing in a common environment, we examined developmental changes in hepatic DNA methylation between the Immediate (age-1) and Persistent (age-2) sample sets. Immediate time-point, but also indicates separation between the Immediate and Persistent time-points. The fish that experienced early rearing in the stream cannot be distinguished from those that experienced early rearing in the hatchery at the Persistent time-point. The second principal component, accounting for 1.6% of the variation further separates the Immediate and Persistent time-point. DNA methylation patterns displayed global significance between the rearing groups and time-points We performed a PCA on the DNA methylation data from the Immediate (age-1) and Persistent (age-2) samples using rearing environment as a model covariate and identified 25,513 DMCs  are associated with genes  samples, but these DMRs were the most extreme in terms of having some of the largest absolute differences in methylation. For example, Developmental DMRs are reported in There were 403 DMRs identified. Only 63 DMRs were hypomethylated in We performed DNA methylation analysis on sperm of fish that experienced early rearing in either the hatchery or stream environment, but sexually matured in a common environment, to test for the potential intergenerational effects of rearing environment on DNA methylation patterns. A similar proportion of males from the hatchery and stream rearing group, 18% and 20% respectively, matured at age-2. RRBS was performed on 30 males from each group. Nineteen families were represented within this sample set; eight families were represented in both hatchery and stream treatments. . Approximately half of the DMCs were hypomethylated in fish that experienced early-rearing in the hatchery environment. Fourteen of the DMCs were associated with genes . The DMCImmediate and Persistent datasets. There were no overlaps with DMCs identified in the Immediate sample set, but one sperm DMC, that made up a larger DMR, was within 60 bp of a DMC identified in the Persistent sample set. These proximal DMCs, taken from different tissues at the same time-point, were both hypomethylated in fish that had an early hatchery-rearing environment. These CG sites are located on Chromosome 24, approximately 3 kb upstream of a Heat shock cognate 70 kDa protein gene.We evaluated the 19 DMC in sperm to determine if any of these CG sites were also differentially methylated in the liver of the per se, they may be reflecting how physiological responses at this later time-point may have been shaped by the early environment indirectly. Small differences observed in the sperm methylome may suggest limited potential for intergenerational changes, but care should be taken in this interpretation, as only a fraction of the genome is analyzed in RRBS and the high degree of methylation in this cell-type makes small changes difficult to detect. The effect of family observed in the DNA methylation patterns in sperm were striking and highlight the importance of accounting for relatedness in studies of DNA methylation in natural populations.To minimize the loss of fitness phenotypes that have been observed in hatchery-reared steelhead, a better understanding of how the hatchery environment is affecting subsequent life-history and fitness traits as well as the underlying mechanisms is needed. Here we used a lab-scale study to investigate the dynamics of hatchery-induced DNA methylation changes in fish with known genetic background. The aim of our study was to investigate the potential of DNA methylation to ‘store’ information about the hatchery-environment in the form of an epigenetic memory. This is the first study to examine the effects of captive rearing across life stages and tissues in fish with known relatedness. Results suggest that initially DNA methylation changes in response to hatchery-rearing are substantial, but that developmental and environmental signals continue to shape the methylome, in a sense “overwriting” the original signal of the hatchery environment. Nevertheless, there are differences in DNA methylation patterns between hatchery- versus stream-reared fish after a year of common rearing. While these differences are not “persistent” TATA-binding protein-associated factor 172 and DnaJ homolog subfamily C member 17, were both found to be hypermethylated in hatchery-reared juveniles, perhaps suggesting methylation patterns associated with these genes may be more general responses to hatchery rearing in juveniles, while methylation changes in other regions may be more hatchery, tissue or species-specific. TATA-binding protein-associated factors are associated with activating RNA polymerase III as part of a complex with TATA-binding proteins [DnaJ homolog subfamily C member 21, was shown to be differentially regulated in the offspring of hatchery versus wild steelhead that had been reared in a common environment [AKT serine/threonine kinase 1 and protein kinase C alpha) and genes capable of binding EGF . Although additional research would be required to understand the direct relationship between DNA methylation changes and gene expression changes in the current study, these results highlight biological pathways, such as those related to liver development and injury, may be impacted by hatchery rearing.We identified substantial DNA methylation differences in the livers of juvenile male steelhead that had experienced a majority of their early rearing in a hatchery compared to stream environment. Global analyses of the liver methylome showed separation between the hatchery- and stream-reared fish when sampled after 8 months of rearing in the two environments . Our resproteins , while Dproteins . A gene ironment  perhaps ironment . DiffereIn many salmon and steelhead hatcheries, environmental conditions in the hatchery are manipulated in order to accelerate growth. In particular, temperature and diet/feeding rate during early life-history can be very different than environmental conditions experienced by their wild conspecifics ,58. TherPersistent sample contrast) no longer distinguished hatchery versus stream-reared individuals as they did in the age-1  males. This is consistent with a previous study that also failed to identify global differences in DNA methylation between adult hatchery and natural-origin steelhead [Persistent and Immediate time-points. The DMCs we identified in livers after a year of common rearing were not “persistent” per se as we found a vast majority of the DMCs identified were unique compared to those identified in the Immediate sample contrast. It appears that although the vast majority DNA methylation changes observed immediately post treatment were reversible, a signal of early-rearing environment was still present in the DNA methylation patterns of the liver in age-2 fish, albeit at new, or not previously identified, genomic loci. This suggests that the early rearing environment affected the response to the subsequent rearing environment, but a greater understating of molecular mechanisms underlying how the early-environment continues to influence the dynamic DNA methylation landscape over developmental time is still needed.Temporal stability of environmentally-induced DNA methylation programming is an often-overlooked area of study. In this study, we tested whether hatchery-induced DNA methylation differences were reversible or persistent by analyzing methylation in the liver of hatchery- and stream-reared males after a year in a common environment. Only sexually immature males were used to avoid confounding effects of maturation. Hierarchical clustering of global DNA methylation levels in the liver of age-2 males  and age-2 (Persistent), and of those, only a single locus, associated with the gene Dystrophin, showed a consistent direction of methylation change (hypomethylated in hatchery- versus stream-reared fish) at both time-points. Dystrophin has been shown to be differentially expressed in liver of trout fed an animal- versus a plant-based diet [Persistent sample contrast could be evidence that early differences in diet  between hatchery and stream-reared fish are being reflected in the liver even after a year of common rearing.Despite the fact that over 70% of the DMC identified in livers of the males in the e-points . We acknsed diet . In mammsed diet . One couThis experiment was designed to mimic the early-life conditions experienced by hatchery- and wild-reared steelhead; however, due to capacity restraints on the simulated stream, once the rearing treatment was complete, just 2 months prior to the time when hatchery steelhead are released into the wild, fish from both treatments were placed into tanks for a year of common rearing. It is likely that DNA methylation patterns observed in the liver after one year in the tank environment on artificial feed reflect transcriptional and physiological responses to diet, temperature, water chemistry, and density of the fish in the tank among other factors. While the methylation profiles in the age-2 fish are likely not reflective of DNA methylation patterns of similarly aged steelhead in the wild, the biological function of differentially methylated genes at this time-point is applicable to investigations into nutritional and other types of developmental programming in aquaculture. For example, controlled stress exposure in early-life may promote growth in Atlantic salmon ,67, and Persistent time-point may be reflecting greater overall variation among individuals in DNA methylation. Indeed, there is clearly larger variation in liver DNA methylation patterns among the Persistent (age-2) compared to the Immediate (age-1) individuals according to principal component analysis (Immediate) and age-2  males directly.In general, the data from the present study support the notion of plasticity over persistence in DNA methylation differences induced by hatchery rearing, however, this does not tell the whole story as new DMCs were identified at this time-point. The lack of differentiation between the hatchery and stream-reared fish at the analysis . This maPersistent) and age-1  immature male steelhead, could be associated with smoltification. Global methylation analysis using PCA clearly separates age-1 and age-2 individuals  parr and smolts [Enrichment analysis of genes associated with DMCs from the gulation . These cgulation . The livgulation , increasgulation  and Gh [gulation  signalingulation . While ogulation ,95. Althd smolts .While the comparison of DNA methylation between different life stages in steelhead does not directly address questions related to the role of epigenetics in mediating hatchery-induced phenotypes, it does highlight an important aspect of study design and biological interpretation that needs to be considered. This comparison does highlight the temporal plasticity of DNA methylation regardless of environment. DNA methylation appears to strike a balance between stability and plasticity; therefore, inferring the stability of environmentally-induced DNA methylation changes based on a single time-point or life stage may be misleading.cyclic AMP-responsive element-binding protein gene, a transcription factor with known function in spermatogenesis [The period of primordial germ cell differentiation into oogonia or spermatogonia has been shown in mammals to be a period when environmental factors may influence DNA methylation patterns of the germ line, and could potentially be passed to subsequent generations . We hypoogenesis . A singlogenesis  and in cogenesis . While ioocyte Zn finger XICOF20, which encodes a zinc finger protein with a Krüppel-associated box (KRAB) domain, was found to be associated with DMCs in the sperm of hatchery-reared fish in both studies. Zinc finger proteins are transcription factors with functions related to DNA/RNA binding, RNA splicing, and protein-protein interactions [Fewer DMCs and DMRs were identified in sperm in the current study compared to a previous study in Methow River steelhead that identified over 100 DMRs in sperm of wild-caught hatchery versus natural-origin steelhead . The difractions . Zinc firactions . This geThe effect of family on DNA methylation is the most notable result from global methylation analysis in sperm compared to liver. Hierarchical clustering showed very strong relationships between methylation patterns in the sperm of siblings compared to unrelated individuals . This efThis study is the first to assess the effects of hatchery rearing on DNA methylation patterns in fish across developmental time and in multiple tissues. We found a strong effect of rearing environment on DNA methylation patterns in the liver of males at 10 mpf. While majority of the loci affected by the early rearing environment were reversible, the early-rearing environment continued to affect DNA methylation patterns in the liver after a change in environment (1 year of hatchery rearing). Direct comparison of the liver methylation patterns in age-1 versus age-2 fish highlight the plasticity of DNA methylation across developmental time. The largest differences in methylation were between the age-1 and age-2 fish and genes associated with these DMCs appear to at least in part reflect physiological requirements/changes for smoltification. Results in sperm show little effect of early-rearing on DNA methylation patterns, but strong family effects were observed, highlighting the importance in accounting for relatedness in experimental design. The results of this study are consistent with previous work that hatchery rearing does have impacts on DNA methylation, but highlight the fact that temporal and tissue-specific differences are important to understand for interpretation and implications for phenotypes associated with captive rearing practices.


The relatively recent development of methods to stably manipulate the genetic make-up of many parasites has allowed a better understanding of host-parasite interactions, particularly in vivo. In this regard, the use of transgenic parasites can facilitate the study of immunomodulatory mechanisms under in vivo conditions. Therefore, in this review, we specifically highlighted the current developments in the use of transgenic parasites to unravel the host’s immune response to different life-cycle stages of some key parasite species such as Leishmania, Schistosoma, Toxoplasma, Plasmodium and Trypanosome and to some degree, the use of transgenic nematode parasites is also briefly discussed.Parasites comprise diverse and complex organisms, which substantially impact human and animal health. Most parasites have complex life-cycles, and by virtue of co-evolution have developed multifaceted, often life-cycle stage-specific relationships with the immune system of their hosts. The complexity in the biology of many parasites often limits our knowledge of parasite-specific immune responses, to  Today, despite available treatments, more than 2 billion people are suffering from chronic infections with parasites, resulting in considerable morbidity . Chronicin vivo [Toxoplasma, Plasmodium and Leishmania), robust systems were developed based on classical molecular biology methods such as microinjection, chemical transfection and electroporation, resulting in early advances in the understanding of the interactions between the immune system and these parasites. In contrast, for multicellular parasites these methods were not optimal and therefore, these parasites are now manipulated using transduction based on retroviral vectors.Recent progress has been made in the development of transgenic parasites allowing for the structural and functional analysis of specific gene products , by for in vivo . For genSchistosoma mansoni lentivirus transduction was necessary to allow a highly efficient introduction of the foreign DNA [Another breakthrough was the discovery that parasites possess the RNA-dependent gene silencing machinery and hence RNA interference (RNAi) has been applied to specifically downregulate genes, as opposed to producing a completely knocking-out of the gene of interest. For multicellular organisms such as eign DNA . Despiteeign DNA .This review highlights the contributions made by studies on transgenic parasites to the understanding of host-parasite interactions and other applications of genetic manipulations in parasites were specifically omitted.The databases PubMed and Web of Science were searched using keywords, including “transgenesis”, “parasite” and “immune response”. The search was conducted during the period of 2017 and 2018 and all the relevant scientific publications were screened following the procedure illustrated in the diagram below . While these antigen-specific CD8+ T cells did not contribute to protection against the blood stage of Plasmodium infection, they played a key role in the initiation of cerebral malaria (CM) and hence are important in the pathology of this infection [One way of demonstrating protective cell-mediated immune responses is with the help of parasites transfected with putative protective antigens, followed by cell-transfer experiments to confirm the protective immune response. One special case of this approach consists of using antigens for which T cell receptor (TCR) transgenic mice have been generated . These mepitopes , recogniepitopes . Blood snfection . This unModel antigens , such as OVA, are frequently expressed in pathogens to investigate the major determinants and pathways influencing T cell responses. In contrast to pathogen-antigens, model antigens have never been subjected to evolutionary pressure and as a result they are unlikely to have been selected to induce biased immune responses. Thus, studying the immune response to these antigens not only allows for the use of many available reagents, but also facilitates the isolation of effects due to the antigen from these due to the pathogen. However, these model antigens are also artificial and therefore may not necessarily reflect the immune responses against pathogen antigens. Nevertheless, they represent useful tools in furthering our understanding of immune responses associated with parasites.+ and OT-I CD8+ T cells specifically recognize epitopes of OVA in Toxoplasma gondii including strains expressing OVA such as type I strain (expressing RH-OVA) and type II strains (expressing Pru-OVA). By injecting these transgenic parasites into mice, Pru-OVA can activate the DCs to produce cytokines within the draining lymph node and a large population of endogenous OVA-specific CD8+ T cells can be generated. Mice infected with RH-OVA have fewer DCs and OVA-specific CD8+ T cells [Other examples of the use of model antigens are studies showing that OT-II CD4 T cells . The expP. berghei have been developed expressing recombinant proteins either in the cytosol or the parasitophorous vacuole membrane (PVM) to assess whether the sub-cellular location can influence T cell responses. OVA and mCherry conjugated with P. berghei heat-shock proteins such as HSP-70 were expressed in the cytoplasm. However, OVA gene sequence conjugated with the HEP17 (EXP1) promoter showed a low level of expression in the PVM [P. berghei lines induce OVA-specific CD8+ and CD4+ T cell responses, PVM-expressed OVA-HEP17 (EXP1) induced higher proliferation of OT-II and OT-I T cells than cytoplasma-expressed OVA-mCherry-HSP70, suggesting that antigen localisation within the parasite affects T cell recognition. Unconjugated OVA is not expressed in the cytoplasm of the parasite and more importantly here, the location and the degree of expression in the parasite also influence T cell responses [Plasmodium by CD8+ T cells during different life-cycle stages of infection was also described [+ T cell response in vivo [P. berghei showed cross-presentation of the antigen by CD8α+ antigen presenting cells in association with MHC class I and II inducing both CD8+ and CD4+ T cells [The expression of model antigens in different part of the parasite using transgenesis have been frequently used to determine the effect of antigen localisation on the host’s immune recognition. For example,  the PVM . Althougesponses . The recescribed . However in vivo . Further T cells .Plasmodium spp. are not the only parasites for which the location of the antigen affects recognition by T cells. Indeed, Toxoplasma gondii has been engineered to express recombinant OVA either in the cytosol or to be secreted into the parasitophorous vacuole [+ T cells and induce IFN-γ production  NT-OVA was unable to achieve this outcome, possibly because less OVA was available in the phagosomes [L. major transfected with either the secreted or the intracellular form of NT-OVA  Salmonella typhimurium flagellin (FliC), (ii) Neisseria meningitidis (FAM18 strain) porin (PorB) or (iii) T. cruzi paraflagellar rod protein 4 (PAR4) genes have been generated. Not surprisingly, strong innate immune responses were observed when mice were infected with these transgenic parasites and the PAMPs activated innate immune cells and these activated immune cells played an important role in controlling T. cruzi infection [T. cruzi helps the pathogen to survive in the host. These studies also showed that inoculation of exogenous PAMPs with T. cruzi and under continuous expression can induce strong CD8+ T cell responses which ultimately leads to the control and clearance of infection  is engineered to induce protective host immune responses [Leishmania spp. engineered to produce monocyte chemoattractant protein 1 (MCP-1) were less pathogenic in susceptible BALB/c mice in vivo, with significant reduction in lesion size compared to wild-type parasites [The two functionally distinct T helper cell populations, which determine the er cells . Howeveresponses . Indeed,arasites . In addiNippostrongylus brasiliensis was engineered in trypanosomes and these transgenic trypanosomes were able to induce a protective immune response against N. brasiliensis, by altering the cytokine environment. In the same study, immune cells exposed to acetylcholinesterase expressing parasites in vivo showed activation of macrophages, production of high levels of nitric oxide and decreasing arginase activity [In another application, transgenic parasites can also be used as vaccine vehicles. Indeed, one study showed the development of a novel system for the expression of nematode secreted proteins in another parasite such as Trypanosoma musculi, a natural parasite of mice. Furthermore, acetylcholinesterase from activity .In summary, the transgenesis of proteins that improve immunogenicity of the parasite have given us insights not only in how parasites evade the immune system, for example by reducing the amount of danger signals they express but more importantly also in the fundamental mechanisms of immune regulation, particularly the role of key cytokines. Some of these engineered parasites might even be used as vaccine vehicles, although this would require passing considerable regulatory hurdles.P. falciparum lines in which eba-175, eba-181, and eba-140 genes were knocked out by targeted disruption, showed that the EBL and PfRh proteins functionally interact during merozoite invasion. Further evidence showed that PfRh and EBL proteins induced antibodies that potently blocked merozoite invasions and hence these antigens could potentially be used as vaccine candidates [eba genes. However, in some instances such as the MSP-1 protein a knock-out strategy can be unsuccessful [Deletion mutants of parasites have been used to investigate the role of critical molecules in the binding of the parasite to the host cells and the effect of antibodies to these molecules in inhibiting this process. Indeed, studies in human malaria showed that erythrocyte binding-like (EBL) proteins and reticulocyte binding-like (RBL or PfRh) proteins are involved in erythrocyte bindings and are targets of human invasion inhibitory antibodies in addition to being important components of acquired protective immunity . A constndidates , 49. Theccessful , for exa[The ability of parasites to modulate immune responses is often mediated by the production of immunomodulatory molecules. Hence, there is a growing interest in the characterization of the mediators released by the parasites and analysing how such molecules can redirect the host’s immune response . This mi, but alsSchistosoma mansoni, are among the pathogenic organisms that invade diverse organ systems such as the lymphatic system, the gastro-intestinal tract and the vascular system with multiple immunomodulatory lines of attack on the immune system. Co-evolution of parasites and their hosts’ immune system has resulted in a delicate balance that allows chronic infections to be maintained without provoking fatal immunopathology or overwhelming infection in the host [S. mansoni egg proteins including Omega-1, IPSE, and kappa-5 are the main contributors of egg-induced immune regulation [Omega-1, is attributed to an altered interaction of DCs with Th cells and the initiation of mRNA and rRNA degradation. This leads to abolished protein-expression by DCs. These interactions and the immunomodulatory mechanisms can be investigated by downregulating Omega-1 in S. mansoni eggs and studying the effect in vivo [in vitro and in vivo through the conditioning of DCs. Indeed, the depletion of this protein totally abrogates the maturation of T cells induced by schistosome SEA in vitro [S. mansoni eggs leads to the production of IFN-γ [S. mansoni eggs revealed a characteristic Th2 cytokine profile  with low levels of Th1 , and Th17 cytokines . Moreover, intravenous injection of wild-type S. mansoni eggs increased numbers of eosinophils, granulocytes, T helper cells, alveolar APCs and B cells [Parasites, such as the host , 53. S. gulation . The eff in vivo . Omega-1in vitro . As mentof IFN-γ  Fig. 6)Schistoso B cells . In contS. mansoni eggs and the results conformed these discovered by downregulating this gene using the RNAi/lentiviral transduction system, namely a reduction of pulmonary granuloma [In recent years the CRISPR/Cas9-based genome edition tools have become a standard way of disrupting key genes in order to study their effect. However, most of the studies so far have used this technique to investigate the parasite biology rather than host-parasite interactions, which fall outside of the scope of this study. While one can expect many future studies to include CRISPR/Cas9-based genome edition tools in the study of immune responses to parasite, the only study so far targeted Omega-1 of ranuloma .L. mexicana cysteine peptidase B in these parasites resulted in similar lesion-development compared to wild-type parasites in the early stages of infections [-/- parasites cleared the infection after inducing a STAT4 and IL-12-dependent Th1 response, indicating that cysteine peptidase B suppresses the immune response [In another parasite example, deletion of fections . Howeverresponse .6-Cys B gene family, which plays a role during development of sexual stages of Babesia bovis, resulted in inhibiting parasite invasion of red blood cells [Finally, disruption of the conserved od cells . Such noTaken together these experiments demonstrate the usefulness of different gene-disruption and downregulation technologies for the study of gene function of parasites including these highlighted here in relation to host-parasite interactions. While gene disruption offers a more absolute effect because the protein of interest is completely absent, gene downregulation has the advantage that if the gene is essential the parasite might survive although in an impaired state and hence an effect might still be observable in that case.in vivo depletion of specific cells, cytokines and their receptors showed that the immune response to nematodes in a murine model is mainly dependent CD4+ T cells [As outlined in this review, recent advances in transgenesis of complex parasites have allowed significant progress towards understanding the immunobiology of a range of parasites. These advances could potentially also be applied to even larger parasites, such as parasitic nematodes. Indeed, the tracking of nematode-specific T cell responses has so far been a challenge . The use T cells , 62. Par T cells . While t T cells . TherefoWe have reviewed current advances focusing on the use of transgenesis as a tool in the study of host-parasite interactions.in vivo.The transgenesis approach is now becoming a novel technique in the integration of foreign genes reliably into the host genome for functional analysis of the immune response To date, few transgenic parasites have been developed and improvements in the advancement of this field is required to increase our knowledge of parasite immunology.in vivo studies might soon become a practical reality.We foresee that such advancement in transgenesis technology could pave the way to rapid developments in functional genomics as it relates to host-parasite interactions. For example, high throughput insertional mutagenesis, expansion of RNAi for We also expect the advances achieved thus far can be adapted in a wide range of pathogenic parasites to understand their nature of interaction with the host.Importantly, parasites in which one key immune mechanism has been eliminated could potentially reveal additional mechanisms that are normally masked by the overpowering effect of the main immune mechanism. The use of transgenic parasites in studying immune response is summarised in Table in vivo. Indeed, using this versatile technology, it is now possible to unravel the detailed immune response to parasites. While this review highlights how transgenic parasites have influenced our understanding of the host-parasite interaction, many additional questions remain and establishing these advanced transgenesis methods for applications in an ever-increasing variety of parasite species will undoubtedly result in the discovery of fascinating insights about the interactions of parasites with the immune system of their hosts.In conclusion, transgenic parasites have been utilised in a wide range of applications to investigate complex host-parasite interactions