DYNC2H1 variants in a couple whose five fetuses had short-rib polydactyly. Prospective analysis of two couples with multiple pregnancy terminations for fetal akinesia syndrome was performed and a diagnosis was obtained in both the families. The first couple were each heterozygous for a previously reported GLE1 variant, p.Arg569His or p.Val617Met; both were inherited by their two affected fetuses. The second couple were each heterozygous for a novel RYR1 variant, c.14130-2A>G or p.Ser3074Phe; both were inherited by their three affected fetuses but not by their unaffected child. Biallelic GLE1 and RYR1 disease-causing variants have been described in other cases with fetal akinesia syndrome. We conclude that exome sequencing of parental samples can be an effective tool for diagnosing lethal recessive disorders in outbred couples. This permits early prenatal diagnosis in future pregnancies.Rare disorders resulting in prenatal or neonatal death are genetically heterogeneous. For some conditions, affected fetuses can be diagnosed by ultrasound scan, but this is not usually possible until mid-gestation. There is often limited fetal DNA available for investigation. We investigated a strategy for diagnosing autosomal recessive lethal disorders in non-consanguineous pedigrees with multiple affected fetuses. Exome sequencing was performed to identify genes where each parent is heterozygous for a rare non-synonymous-coding or splicing variant. Putative pathogenic variants were tested for cosegregation in affected fetuses and unaffected siblings. In eight couples of European ancestry, we found on average 1.75 genes (range 0–4) where both parents were heterozygous for rare potentially deleterious variants. A proof-of-principle study detected heterozygous  Rare disorders that result in antenatal or neonatal death are both phenotypically and genetically heterogeneous. Diagnosing lethal fetal disorders has previously been very difficult because of the large number of potential genes, the phenotypic variability associated with many known genetic causes and the challenges of defining phenotype and pathology in a mid-gestation fetus. For couples with multiple affected fetuses, most result from autosomal recessive mutations with a 25% recurrence risk for each future pregnancy. Current testing of a limited number of genes guided by phenotype often fails to achieve a molecular genetic diagnosis. Consequently, early prenatal testing is not possible and ultrasound diagnosis in the second trimester is the only potential option.et al1). In outbred families, a novel disease gene can be revealed in a single affected individual through the detection of compound heterozygous variants.3Exome sequencing is a powerful tool for both disease gene identification and diagnosis of monogenic disorders. Autosomal recessive diseases can be investigated by a combination of autozygosity mapping and exome sequencing in consanguineous pedigrees to identify homozygous pathogenic variants  family where five pregnancies were affected with short-rib polydactyly caused by compound heterozygous We then prospectively investigated two unrelated couples who had terminated at least two pregnancies after an ultrasound diagnosis of a lethal disorder. X-linked inheritance was unlikely due to at least one affected fetus being female. The pedigrees and clinical characteristics of the affected fetuses are shown in CHRNG, BIN1 or DNM2. Testing of the mother for acetyl cholinesterase receptor antibodies was negative.The first couple had two fetuses affected with fetal akinesia thought to result from a centronuclear or myotubular myopathy based on post-mortem muscle histopathology. They had no live offspring and were referred for exome sequencing 6 weeks into their third pregnancy. Karyotype and array CGH testing did not detect an abnormality, and previous testing of fetal samples had not identified deleterious variants in CHRNG, CHRND, CHRNA1, RAPSN, DOK7 or SMN1. Exome sequencing was requested during their fourth pregnancy (8/40).The second couple had a healthy daughter and then underwent three pregnancy terminations. The phenotype included joint contractures, pterygia, pulmonary hypoplasia and hydrops. Karyotype and array CGH testing did not detect an abnormality and no deleterious variants had been found in Genomic DNA samples were fragmented and indexed adaptors ligated before hybridization with the Agilent SureSelect All Exon v4 capture kit , designed to target 51 Mb of genomic sequence comprising of 20 965 genes and 334 378 exons from the CCDS, RefSeq and GENCODE databases. Paired-end 100-bp reads were sequenced on a HiSeq 2500 , 6 exomes per flow cell, to generate 80 × mean coverage with >75% of target bases at ≥30 × . Sequencing reads were separated by index, aligned with BWA-MEM to the hg19 reference genome and duplicates removed with Picard. GATK v2.7-2 was used for indel realignment, variant calling by UnifiedGenotyper and variant quality filtering on hard filters as recommended for exome data. We called variants within two pools of samples: one group included the eight healthy couples (16 samples) that we initially used to test the strategy, and the second group included the three affected families (six samples) with multiple pregnancy terminations for a presumed autosomal recessive disorder.Variants were annotated using a Linux version of Alamut-HT-1.1.11, which takes a VCF file as an input and annotates all SNVs and InDels using a range of different variant and genomic databases, including HGMD Professional. Because we were searching for variants causing a very rare lethal disorder, we applied the following criteria to look for rare potentially deleterious variants: MAF<1% in dbSNP137 variants, or MAF<0.01% in ESP or 1000 Genomes, or present in HGMD as a disease-causing variant. We excluded variants identified in 106 in-house control exomes. Variants were restricted to non-synonymous, splice site or within−50/+10 bp of flanking exons and predicted by Alamut-HT to affect splicing. Alamut-HT uses three different tools  in an algorithm that makes an overall prediction of potential splicing effects, namely creation of a new acceptor or donor site, activation of existing cryptic sites or an effect on the nearest splice site. We identified genes where parents either shared the same heterozygous variant (for which offspring could be homozygous) or had different heterozygous variants in the same gene .GLE1 or RYR1 were confirmed in the parents by PCR/Sanger sequencing and tested in their offspring to investigate cosegregation with disease. PCR primers are available on request.Putative deleterious heterozygous variants in www.LOVD.nl/DYNC2H1 ; www.LOVD.nl/RYR1 ; www.LOVD.nl/GLE1 .All confirmed variants were submitted to locus-specific databases within the LOVD 3.0 shared installation: We tested a strategy to diagnose lethal autosomal recessive disorders by exome sequencing in non-consanguineous parents. Analysis of eight unrelated couples of European ancestry showed that their exomes contained an average of 103 rare heterozygous non-synonymous coding or splice-site variants . The number of genes in which rare potentially deleterious heterozygous variants were identified in both members of the couple ranged from 0 to 4 see . The aveDYNC2H1 variants. Rare, potentially deleterious heterozygous variants were identified in both partners for two genes, NR1D1 and DYNC2H1 (DYNC2H1 variants as c.2819-14A>G (NG_016423.1) and c.7577T>G, p. Ile2526Ser (NM_001080463.1). In silico tools (Alamut) suggest that the p. Ile2526Ser missense variant is deleterious, and the c.2819-14A>G variant is predicted to create a new splice acceptor site: 13 bp upstream from exon 20 (numbering according to NG_016423.1).In a proof-of-principle study, we sequenced the exomes of a couple who had five fetuses affected with short-rib polydactyly caused by compound heterozygous  DYNC2H1 . Exome sWe then investigated two families referred to our local clinical genetics service because of a history of multiple fetuses terminated after an antenatal ultrasound scan diagnosis of fetal akinesia syndrome.CLIP1 and GLE1, were found in both partners (GLE1 variant (NM_001499.2) and that both variants were present in the two affected fetuses. These GLE1 variants, p.Arg569His and p.Val617Met, have been reported in Finnish patients with clinical phenotypes consistent with fetal akinesia syndrome.6The first couple had two pregnancies affected with pterygia and/or joint contractures. Rare heterozygous missense variants in two genes, partners . Sanger RYR1 variants (NM_000540.2). All three affected fetuses were found to be compound heterozygotes and the couple's unaffected daughter had not inherited either variant. The c.14130-2A>G variant affects a conserved splice acceptor site, but p.Ser3074Phe is a novel missense variant. Assuming a very rare autosomal recessive disorder, where the probability of affected and unaffected offspring is 0.25 and 0.75, respectively, we calculated the maximal LOD score in the four offspring (three affected and one unaffected) as −log10(0.253 × 0.75)=1.9.The second couple had a healthy daughter followed by three fetuses with arthrogryposis. We identified four genes in which both parents were heterozygous for rare variants with possible pathogenic consequence . Sanger DYNC2H1 variants causing short-rib polydactyly. Two families presenting with a current at-risk pregnancy were then studied prospectively, and a molecular genetic diagnosis was obtained in both families through the identification of GLE1 and RYR1 variants causing a severe form of fetal akinesia syndrome with arthrogryposis.We developed a strategy to diagnose rare autosomal recessive lethal disorders by exome sequencing in non-consanguineous couples with a history of multiple affected fetuses. The aim was to obtain a molecular genetic diagnosis and enable prenatal testing in current/future pregnancies. A proof-of-principle study successfully identified heterozygous GLE1 missense variants have already been reported in separate Finnish families who also carry the Fin(Major) c.432-10A>G variant that creates an illegitimate splice acceptor site; one patient compound heterozygous for p.Arg569His and Fin(Major) was affected with lethal congenital contracture syndrome type 1 (OMIM 253310), while six patients compound heterozygous for p.Val617Met and Fin(Major) had lethal arthrogryposis with anterior horn disease (OMIM 611890).6 Interestingly, the frequency of Fin(Major) is up to 2% in Finnish sub-populations,6 while p.Arg569His (rs121434407) has frequency of 0.03% in the ESP database commonly used for variant frequency filtering, owing to the large number of Finnish samples contributing to this project. We conclude that compound heterozygosity for these known GLE1 variants is the cause of fetal akinesia syndrome in this family.The two RYR1 gene are a known cause of susceptibility to malignant hyperthermia.7 There is no history of malignant hyperthermia in either the parents or wider members of family 2 but penetrance of this condition is known to be low. Dominant or recessive RYR1 mutations are a common cause of congenital myopathies.9 There is considerable phenotypic variability reported and a small subset have a more severe form characterized by a fetal akinesia syndrome,11 including one fetus with compound heterozygous RYR1 missense variants that was terminated at 32 weeks' gestation.10 Cosegregation of the c.14130-2A>G and p.Ser3074Phe RYR1 variants in family 2 is consistent with linkage . We conclude that these RYR1 variants are highly likely to be the cause of fetal akinesia, arthrogryposis and pulmonary hypoplasia in all three fetuses.Heterozygous variants in the RYR1 variants were confirmed shortly before this scan and they were offered the option of prenatal testing by amniocentesis sampling. In view of the normal ultrasound scan, they decided not to risk a miscarriage by undergoing an invasive procedure. Both couples have indicated that they would seek early prenatal testing in future pregnancies.Ultrasound analysis at 20 weeks for the on-going pregnancies showed no evidence of fetal akinesia syndrome. Couple 2's 13 We considered the alternative approach of sequencing the exome of one of the fetal DNA samples, but there is a higher risk of exome sequencing failure as the quality of DNA extracted from terminated fetuses can be poor compared with leukocyte DNA from healthy adults, particularly if the only source of fetal DNA is formalin-fixed paraffin-embedded tissue. In addition, the amount of fetal DNA available may be insufficient for exome sequencing or limited in quantity such that it is prudent to sequence the parents and save the precious fetal sample for confirming potential pathogenic variants or for future studies. For the couple with RYR1 variants, cell culture was required to obtain DNA for cosegregation studies in two of the fetuses. This took 3 weeks, by which time the potentially pathogenic variants had been identified through analysis of the parental exome sequence data.Our strategy of exome sequence analysis in the parents of affected fetuses generated a very small number of potential pathogenic variants for confirmation and cosegregation analysis by Sanger sequencing. Several studies have diagnosed recessive disease through the identification of compound heterozygous variants by exome sequencing in a single individual.We conclude that exome sequencing in non-consanguineous couples can provide an effective means of making a genetic diagnosis of lethal autosomal recessive disorders. Diagnosing lethal fetal disorders has previously been very difficult because of the large number of potential genes, the phenotypic variability associated with many known genetic causes and the challenges of defining phenotype and pathology in a mid-gestation fetus. Sequencing parental samples overcomes issues of limited quality or quantity of fetal samples. A genetic diagnosis confirms the risk for future offspring and permits early prenatal diagnosis or preimplantation genetic diagnosis in future pregnancies. This in turn reduces the anxiety associated with waiting until mid pregnancy for an ultrasound diagnosis, and avoids the added distress of a late termination of pregnancy. The strategy is also applicable to those disorders not detectable by ultrasound diagnosis where late fetal demise or a neonatal death could not otherwise be predicted.


However, staining procedures and discontinuous monitoring are major drawbacks hampering accurate assessment of wound assays. We therefore investigated digital holographic microscopy (DHM) to appropriately monitor wound healing in vitro and secondly, to provide multimodal quantitative information on morphological and functional cell alterations as well as on motility changes upon cytokine stimulation. Wound closure as reflected by proliferation and migration of Caco-2 cells in wound healing assays was studied and assessed in time-lapse series for 40 h in the presence of stimulating epidermal growth factor (EGF) and inhibiting mitomycin c. Therefore, digital holograms were recorded continuously every thirty minutes. Morphological changes including cell thickness, dry mass and tissue density were analyzed by data from quantitative digital holographic phase microscopy. Stimulation of Caco-2 cells with EGF or mitomycin c resulted in significant morphological changes during wound healing compared to control cells. In conclusion, DHM allows accurate, stain-free and continuous multimodal quantitative monitoring of wound healing in vitro and could be a promising new technique for assessment of wound healing.Impaired epithelial wound healing has significant pathophysiological implications in several conditions including gastrointestinal ulcers, anastomotic leakage and venous or diabetic skin ulcers. Promising drug candidates for accelerating wound closure are commonly evaluated in  Vice versa, impaired wound healing has a tremendous pathophysiological implications in several conditions such as gastrointestinal ulcers in vivo models can be complex and of limited availability in vitro wound assays, such as the classical scratch assay established by Burk et al.in vitroCell layer wound assays’) that are separated by a 500 µm wall, which on removal leaves a well-defined border Epithelial wound healing is a common physiological process. In particular, within the gastrointestinal tract, there is persistent regeneration of epithelial cells to compensate physiological exfoliation of surface cells in vitroin vitro has been reported by Keese et al. Recently, bright field images and Zernike phase contrast images recorded with time-lapse video microscopy were established for analysis of wound healing assays Digital holographic microscopy (DHM), a variant of quantitative phase microscopy, enables not only stain-free quantitative phase contrast imaging but also assessment of cell thickness and tissue density by measuring optical path length delay in vitro in a stain-free and continuous manner and to test its properties for quantitative determination of cellular changes upon cytokine stimulation. To the best of our knowledge, this is the first time a multi-parameter analysis of cellular growth and motility from quantitative phase images during epithelial wound healing has been performed.The aim of this study was to evaluate DHM as a novel method to accurately assess wound healing in vitro, Caco-2 cell wound assays were analyzed (see Cell layer wound assays’) and results compared to microscope images acquired by white light illumination. Mimicking different physiological situations, cells were stimulated with either epidermal growth factor  were analyzed as described in the section ‘Determination of the cellular refractive index and the cell volume’.Zytotoxic agents such as mitomycin c can significantly impair epithelial migration and proliferation without affecting protein biosynthesis controlr = 7.2±1.2 µm was obtained .After stimulation with EGF, the refractive index of the treated cells was comparable to the unstimulated  pg vs. EGFDM = 542±27 pg; P<0.001; Finally, the cellular dry mass was determined as described in section “Taken together, DHM was not only able to quantify morphological characteristics like volume and cell density and dry mass of Caco-2 cells but could additionally reveal alterations of these features upon stimulating and inhibiting cytokine treatment as compared to untreated control cells.Analysis of cellular growth and thickness with quantitative phase microscopy”. Due to the spatial heterogeneous growth behavior of the Caco-2 cells  and mediumn = 1.339. For control cells, the increase of V of the cell layers, the absolute data for the area cS covered by the cells in 3 at the end of experiment. In correspondence to 3 vs. 97994 µm3). The similar increase of cS and cell volume for EGF stimulated cells and control cells may be explained by inhibitory effects of mitomycin c on the EFG stimulated cells, as stimulated and inhibited cells were observed in a single assay with the same cell culture medium.To assess the impact and the duration of stimulating agents on epithelial wound healing, continuously monitoring of the wound closure area would be desirable. Simultaneous determination of changes in cellular dry mass and volume may give additional insights into cell cycle related processes such as cell division and cell viability ells see  the achiperiment . AlteredFinally, we estimated the total number of cells in the wound gap by dividing the relative dry mass  through 2/min. A significantly enhanced area increase after EGF stimulation as compared to mitomycin c treatment was observed  and EGF-stimulated cells (right) from a single representative single experiment. Upon stimulation with EGF, cellular thickness of Caco-2 cells was markedly increased as compared to mitomycin c-treated cells. Finally, DHM examinations also allowed assessing spatial changes in cellular thickness during the wound healing process. in vitro and to provide simultaneous quantification of key cellular characteristics such as cell volume, cell thickness, dry mass and cell density which may help to characterize therapeutic effects of potential drug candidates.In this study, we prove DHM to enable continuous, stain-free monitoring of intestinal epithelial wound healing in vitro is traditionally performed by the use of mechanically induced wounds and healing assessed by the number of cells beyond the wound edge et al. demonstrating that human osteosarcoma U2OS cells double their dry mass before entering mitosis and detecting that daughter cells possess exactly one half of the parental cell mass Proliferation and migration are two major steps required for successful wound closure following ulceration and inflammation in vitro is not provided by traditional approaches.In There is evidence, that wound healing is significantly influenced by the environment, surrounding cells and tissues. Recently, it was demonstrated that deformation and extracellular pressure can stimulate intracellular enzyme activity et al. demonstrated that DHM may also be used to differentiate between apoptosis and necrosis by assessment of cellular volume in vitroet al. demonstrated DHM to be ready to use for assessment of cytotoxicity and cell viability by determination of morphological and biomolecule (protein and nucleic acid) changes. The authors additionally report DHM to be a magnitude faster than automated standard fluorescence microscopy Recently, Pavillon It should be mentioned that our results revealed that EGF-stimulated Caco-2 cells grow with a similar dry mass rate like untreated control cells but show different morphological features which is indicated by a significant difference in the cell layer thickness. These findings may be explained by the specific design of our experiments in which EGF and mitomycin c were applied in a single assay. Nevertheless, the obtained results also demonstrate that the accuracy of the applied DHM method is sufficient to detect, for example interference of different cytokine in an assay.in vitro. Although the detection of individual cells is limited as successful identification depends strongly on the morphological properties of the individual cell type, DHM can continuously quantify minimally invasive proliferation and migration of an ensemble with only low exposure of the sample to light. Furthermore, DHM is able to distinguish between both processes by biophysical information that is retrieved by optical path length changes without molecular markers. In addition, DHM can detect cellular hypertrophy as well as atrophy and due to the working principle of DHM, direct monitoring of cell motility and simultaneous determination of their key morphological characteristics can be performed. Moreover, the use of DHM may not be limited to monitoring of mammalian cells but was has already been shown feasible for assessment of bacteria In summary, our results demonstrate that DHM provides several advantages with regard to previously established methods for monitoring of wound healing in vitro assays using label-free quantifying proliferation and migration with biophysical parameters. As DHM is able to simultaneously assess cellular characteristics by continuously monitoring and quantifying cell migration, morphological alterations and proliferation, DHM can assist in the evaluation of potential therapeutics, help elucidate the specific role of certain cytokines for wound healing, and help dissect cellular alterations which may be related to distinct cellular functions, enabling investigators to perform automated, cost-efficient and minimally invasive quantitative assays with minimized sample interaction in a flexible and more sophisticated way.In conclusion, we propose that the above-presented parameters give significantly advanced insights into cellular characteristics during wound healing in 2, 95% humidity environment at 37°C. Cells were seeded on ibidi dishes (35 mm with high culture-insert coating) at a density of 4×105 cells/cm2 for wound healing assay. Three days after seeding, the medium was changed every third day. Experiments were conducted on the 4th day of culture.Caco-2 cells (passages 22-28)  were cultured in Dulbecco's Modified Eagle Medium  supplemented with 20% foetal bovine serum , 1% non-essential amino acids and 1% penicillin/streptomycin in a 5% COIn vitro wound assays were performed using IBIDI Culture-Inserts according to Shih et al. in vitro wound healing assays with DHM, the cell culture medium was replaced by HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffered medium (20 mM HEPES) and Caco cells were observed in time-lapse series for 40 h with the experimental setup described in section ‘Quantitative phase imaging with digital holographic microscopy’. Digital holograms were recorded every 30 min. The obtained quantitative DHM phase contrast images were further evaluated to quantify cell proliferation and migration as described in section ‘Cell proliferation rates during wound healing’. Experiments were repeated in triplicate as a minimum.λ = 532 nm, Coherent Compass 315M, Coherent, Luebeck, Germany). The cell cultures were observed in special Petri dishes for wound healing observations . The sample was illuminated with laser light in transmission (object wave) and imaged by a microscope lens and a tube lens on a charge-coupled device camera . The object wave was superimposed with the slightly tilted reference wave for the generation of off-axis holograms which were recorded by the camera sensor and transferred to an image-processing system using custom built C++ based hologram acquisition software. For imaging of adherent cells during wound healing a 10× microscope lens  was utilized while suspended cells were observed with a 20× microscope lens .An inverted microscope  with an attached DHM module based on a principle described in Δφ depends on the cell thickness d, the integral cellular refractive index celln, the refractive index mediumn of the cell culture medium λ of the laser light used in the DHM system:The reconstruction of the digitally captured holograms was performed by spatial phase shifting reconstruction in combination with optional holographic autofocusing as described with details elsewhere  can be related to the cellular response to drugs. Thus, celln and V were analyzed for inhibited and stimulated Caco-2 cells as well as for untreated control cells. For determination of celln and V, cells were detached (trypsinized) and suspended in cell culture medium with an osmolality of 320 mOsmol/kg in petri dishes . For each sample, digital holograms of N = 89 selected single cells with spherical appearance were recorded. From the resulting quantitative DHM phase contrast images, the integral cellular refractive index celln and V were determined by numerical fitting of Eq. (1) as described with details previously The determination of the cell thickness with Eq. 1 requires information about the integral cellular refractive index cS covered by the cells was determined in quantitative DHM phase images by image segmentation using the free software cell profiler  the average thickness Furthermore, from P-value <0.05 was considered to be statistically significant.Data were analyzed using SPSS 20.0 (IBM). Results are expressed as means ± standard error. Using the Mann-Whitney U-test and the χ-squared test appropriate comparisons between different data sets were performed. Table S1Numerical data of suspended CaCo-2 single cells displayed in .(DOCX)Click here for additional data file.Table S2Numerical data of temporal relations displayed in .(DOCX)Click here for additional data file.Video S1Movie of segmented quantitative DHM phase images during epithelial wound healing in vitro. Left: CaCo-2 cells inhibited with mitomycin c, right: CaCo-2 cells stimulated with epidermal growth factor (EGF).(MP4)Click here for additional data file.Video S2False color-coded pseudo 3D images of representative quantitative DHM phase contrast images during epithelial wound healing in vitro. Left: CaCo-2 cells inhibited with mitomycin c, right: CaCo-2 cells stimulated with epidermal growth factor (EGF).(WMV)Click here for additional data file.


Panthera tigris) conservation their survival is threatened by poaching, habitat loss and isolation. Currently about 3,000 wild tigers persist in small fragmented populations within seven percent of their historic range. Identifying and securing habitat linkages that connect source populations for maintaining landscape-level gene flow is an important long-term conservation strategy for endangered carnivores. However, habitat corridors that link regional tiger populations are often lost to development projects due to lack of objective evidence on their importance. Here, we use individual based genetic analysis in combination with landscape permeability models to identify and prioritize movement corridors across seven tiger populations within the Central Indian Landscape. By using a panel of 11 microsatellites we identified 169 individual tigers from 587 scat and 17 tissue samples. We detected four genetic clusters within Central India with limited gene flow among three of them. Bayesian and likelihood analyses identified 17 tigers as having recent immigrant ancestry. Spatially explicit tiger occupancy obtained from extensive landscape-scale surveys across 76,913 km2 of forest habitat was found to be only 21,290 km2. After accounting for detection bias, the covariates that best explained tiger occupancy were large, remote, dense forest patches; large ungulate abundance, and low human footprint. We used tiger occupancy probability to parameterize habitat permeability for modeling habitat linkages using least-cost and circuit theory pathway analyses. Pairwise genetic differences (FST) between populations were better explained by modeled linkage costs  compared to Euclidean distances, which was in consonance with observed habitat fragmentation. The results of our study highlight that many corridors may still be functional as there is evidence of contemporary migration. Conservation efforts should provide legal status to corridors, use smart green infrastructure to mitigate development impacts, and restore habitats where connectivity has been lost.Even with global support for tiger ( By virtue of being at the top of the food chain, large carnivores occur at low densities, have large home ranges and therefore require vast areas to harbor viable populations Panthera tigris) acts as a flagship species for the conservation of forested ecosystems throughout its range in Asia c. 200 years ago, and habitat contiguity that permitted genetic exchange between the various regional tiger populations in the area The tiger , with tiger densities ranging from about 0.1 to 20 individuals per 100 km2Currently in India, the once contiguous tiger population is now fragmented with source populations primarily restricted to tiger reserves. A tiger reserve is legally mandated to designate a critical core area wherein human habitation and resource extraction is not permitted  within 185,100 km2 of Central India. Based on our understanding of tiger ecology, we predicted a priori that tigers should occur in vast, undisturbed, productive forest patches, with high density of large wild ungulate prey, which would be negatively impacted by human disturbances a priori hypotheses by spatially explicit modeling of tiger occupancy that accounted for imperfect detections, using covariates obtained by remote sensing and ground surveys covering all forest patches within our study area. We then used this spatially explicit information of tiger occupancy as a resource selection probability function We extensively and intensively collected scat and a few tissue samples across seven tiger reserves in the Central Indian Landscape and first identify tiger individuals by genotyping the DNA extracts using eleven autosomal microsatellite loci. Next we assessed spatial genetic structuring and gene flow in the identified individuals through individual clustering methods. We use likelihood based The majority of field sampling was conducted non-invasively from tiger scat, without animal capture and handling. Permits for collection of tiger scat samples were obtained from the National Tiger Conservation Authority and the State Forest Departments. Capture and radio collaring of tigers required the approval of the Ministry of Environment and Forests, Government of India and the Chief Wildlife Warden, Madhya Pradesh State, under the Wildlife (Protection) Act 1972. The permissions define the conditions required for capture of tigers, which include an approved protocol and participation by a Park Official and supervision by a qualified veterinarian in the capture and collaring exercise. Both these permissions were obtained and strictly adhered to. Capture operations were conducted by trained veterinarians and wildlife biologists as per the protocols of the Wildlife Institute of India and the National Tiger Conservation Authority. A tiger tissue sample was obtained from Satpura Tiger Reserve where the tiger died due to natural causes . This research project was conceived and radio collaring reported in this paper was done prior to the formation of an animal ethics committee at the Wildlife Institute of India.2 (20.1–23.5°N and 76.5–81.5°E) covering the seven tiger reserves of Melghat, Satpura, Pench, Kanha, Tadoba, Achanakmar, and Bandhavgarh along with their buffer zones, corridor habitats and adjoining forested habitats were sampled. Sampled sites covered different types of tiger habitats found in Central India ranging from the tropical moist-deciduous Sal (Shorea robusta) forests in Kanha and Bandhavgarh to tropical dry-deciduous teak (Tectona grandis) dominated forests in Pench, Tadoba and Melghat. The topography varied from about 200 meters above sea level (m a.s.l.) in the low-lying hills to the dadar plateaus and meadows in Kanha (500 m a.s.l.) and the rugged Satpura ranges . The rainfall, primarily restricted to the monsoon season (late June to September end) ranged between 1,000 to 2,200 mm per year. The large mammal fauna found in the region included tigers (Panthera tigris), leopards (P. pardus), sloth bears (Melursus ursinus), dholes , gaurs (Bos gaurus), wild pigs (Sus scrofa), sambar deer (Rusa unicolor), chital deer (Axis axis), barking deer (Muntiacus muntjak), swamp deer (Rucervus duvaucelii) and nilgai antelopes (Boselaphus tragocamelus).The present study was carried out in the global priority tiger conservation landscape of Central India within the states of Madhya Pradesh, Maharashtra, and Chhattisgarh . A foresBlood from 16 radio-collared tigers, one tissue sample from a dead individual and 587 putative tiger scat (faeces) samples were collected between 2006 and 2011 from the seven Tiger Reserves, and at a few intervening forest corridors in the area . The numBamHI restriction enzyme digestion of the mitochondrial DNA cytochrome b gene  statistic Identity analysis option in CERVUS 3.0 FSTRSThttp://cran.r-project.org).In order to investigate the power of the eleven microsatellite loci to distinguish among closely related individuals in the same population, the conservative sibling probability of identity (PhiPT genetic distances www.ncss.com) to understand broad spatial patterns of populations structure in the landscape. Next, we used the Bayesian individual clustering approach in STRUCTURE 2.3.3 K) based on the multilocus genotype data alone. The clustering process ensures that for identified population clusters, deviations from Hardy Weinberg and linkage equilibrium are minimized. We analyzed our data in STRUCTURE by using the admixed model and correlated allele frequencies option to carry out thirty independent simulations at each K (K = 1 to 10), with a burn-in length of one million Monte Carlo Markov Chain (MCMC) steps and data collection phase of five million MCMC iterations. These run times were sufficient to ensure convergence of the Markov chains, and all runs were carried out both with (locprior  = 1) and without (locprior  = 0) using prior sampling locality information. The true K or most likely number of population clusters in the dataset was inferred from (i) the ad hoc parameter of log-likelihood change in probability of individual assignments to K clusters (Ln P(K), K values  and among subpopulations within each group.We used two types of individual-based analyses to assess genetic differences among individuals and assignment patterns of tigers to populations. First, a Principal Coordinates analysis (PCoA) based on pair-wise delta K, . Both thdelta K,  in GENALa priori designation of the migration rate (MIGPRIOR  = 0.05). All other parameters and run times followed previous population clustering runs . We detected no biases in a priori assignment of the migration rate as the selection of particular MIGPRIOR values (0.001 to 0.1) did not substantially influence the STRUCTURE output, therefore only results for MIGPRIOR  = 0.05 are presented here.We used three Bayesian approaches implemented in STRUCTURE 2.3.3 h/LmaxL in combination with the resampling algorithm of Paetkau et al.et al.h/LmaxL, is the ratio of hL, the likelihood of a given individual being assigned to its sampled population to maxL, the greatest likelihood among all sampled populations et al.α levels of 0.01 and 0.05) of h/LmaxL. We considered a minimum log likelihood h/LmaxL ratio of 2.0, which corresponds to a 100 times probability of being cross-assigned, as the threshold level for determining putative migrant status of an individual Next, we used the likelihood-based estimator Third, the non-equilibrium Bayesian assignment test of BAYESASS was used to trace each individual's immigrant ancestry within the last two to three generations. Unlike STRUCTURE and GENECLASS analyses which require loci to follow Hardy-Weinberg and linkage equilibrium, BAYESASS is robust to violations from Hardy-Weinberg equilibrium as it measures contemporary gene flow within the last few generations based only on multilocus allele sharing among individuals Lastly, to corroborate the results of the above assignment based migrant decisions, a likelihood based parentage analysis was carried out in CERVUS 3.0 P<0.01, Lh/Lmax ≥2.0); (ii) observance of>50% migrant or cross-assignment probability in STRUCTURE and BAYESASS; (iii) high assignment probability to first generation immigrant ancestry state (gen1>50%) in both STRUCTURE and BAYESASS; and (iv) high membership (Q>0.8) to a single non-home cluster in the STRUCTURE analysis without prior population information. Further, in most cases of putative migrants, the successful parentage assignment corroborated the migrant status of the individual tiger. We considered a conservative approach by identifying individuals as putative migrants only if all three programs suggested evidence of immigrant ancestry.Designation of migrant status to an individual was contingent upon - (i) significant assignment of first generation migrant status in GENECLASS (m) can be inferred. In BAYESASS, a Bayesian approach incorporating an MCMC sampling scheme is used to estimate migration rates between pairs of populations over the last few (approximately <5) generations back. With an estimated generation time of four to five years in tigers Neθ  and asymmetric mutation-scaled immigration rate M (m/µ). The mutation-scaled immigration rate M, which is the immigration rate m divided by the mutation rate µ, is a measure of the importance of immigration events over mutation in contributing to variation in the population emN, is the product of theta and M. MIGRATE assumes mutation-migration-drift equilibrium with values of M and theta constant over time and parameter estimates in MIGRATE date back nearly 4Ne generations into the past (approximately thousands of years ago). Hence, these migration rates provide estimates of gene flow that post and pre-date the estimated time (approximately 600 years ago) when humans began to significantly alter the habitats in which these tigers currently live.In order to study gene flow across different timescales, we used the programs BAYESASS 1.3 6 MCMC iterations were carried out, by discarding the first 2×106 steps as burn-in and sampling at every 2,000 iteration intervals of the remaining 6×106 MCMC chain. Runs were carried out with a migration rate prior of 0.05 while other parameters were kept at default settings. The average result from three independent BAYESASS runs is presented. Estimates of historical gene flow and effective population size were carried out in MIGRATE by using the Bayesian approach and the Brownian motion model as an approximation for the step-wise microsatellite mutation model. Following initial trial runs, the Bayesian search criteria for the MCMC sampler was set at 10 replicates of one long chain of 50,000 steps with every 100 steps of the chain being recorded, producing a total of 5×107 visited parameter values. The initial 107 steps of the MCMC run were discarded as burn-in, and the remaining 4×107 runs were sampled. To increase efficiency of the sampler, we used four chain-heating temperatures of 1, 1.5, 3 and 10,000, which allows a more efficient exploration of the genealogy space. We used parameter estimates from the initial run to calculate starting values of theta, for use as new parameters during subsequent runs. Parameter estimates from the final run were similar to the results of the initial runs. All MIGRATE runs were carried out on the Bioportal cluster computing facility at the University of Oslo, Norway .For both the BAYESASS and MIGRATE runs, we used the STRUCTURE defined population clusters to estimate pairwise migration rates. BAYESASS runs were performed as described in the preceding methods section on detecting migrants. A total of 8×10M ratio test eN generations in the past. The mode-shift test is more suited for detecting bottlenecks within the last few dozen generations et al.P-values from the Wilcoxon's test were used to examine bottlenecks at each timescale and were assessed at the alpha 0.05 level. The M ratio (M = k/r+1) was calculated from the mean ratio of the number of alleles (k) and the allele size range (r). Assuming loci follow a generalized stepwise mutation model, the loss of rare alleles would diminish the value of k at a faster rate than r thereby a drop in the M ratio below a threshold of 0.68 would be suggestive of populations that experienced a recent bottleneck To detect past occurrences of genetic bottleneck in the sampled populations, we evaluated three summary statistics - (i) Wilcoxon's sign rank test and (ii) mode-shift test, implemented in the program BOTTLENECK 1.2.02 2 of habitat. Surveys were not random, but instead conducted along features that were likely to have tiger sign  so as to maximize detections et al.The entire study area was divided into 10×10 km grids. Each grid that contained potential tiger habitat  was surveyed by replicate search paths for tiger sign. The number of surveys per grid ranged from 3 to 35, and was proportional to the amount of tiger habitat within each grid. Areas under agriculture, industry, and human habitation that were known to be non-habitat for tigers were not surveyed in an occupancy framework. Each survey consisted of a 5 km search for tiger signs with approximately one survey for every five km2 grid ranged from 1 to 24, and were proportional to the quantum of tiger habitat within that grid. The total effort invested in transect surveys was 71,468 km of walking on 26,688 occasions.Within each forest beat, one or two permanent line transects of two to four km in length were delineated. Each transect was walked on two or three subsequent mornings (06:30 to 08:30 hrs) by two observers to record encounter rates of wild ungulates and domestic livestock. Data on number of each species seen and the length of transect were recorded to compute encounter rates of each species. In disturbed forests (outside of protected areas) wild ungulate densities were low, animals were shy, and difficult to record using line transects. Therefore, at every 400 m along the line transect we also sampled a plot of 20×2 m to record ungulate dung. Dung was visually distinguished to species 2 grid ranged from 5 to 120. The total number of plots sampled across the landscape was 51,073.At every 400 m along transects established for ungulate assessment a plot of 15 m radius was sampled to assess indices of human impact. Presence of (a) human/livestock trails within the plot, and (b) sighting of humans and livestock from the plot were recorded 2 Advanced Very High Resolution Radiometer (AVHRR) data, acquired from the National Aeronautics and Space Administration's (NASA) Television Infrared Observation Satellite (TIROS) . Road and drainage information were obtained from Digital Chart of the World . Protected area shape files were obtained from the wildlife database at the Wildlife Institute of India, National Tiger Conservation Authority and State Forest Departments of India. The Shuttle Radar Topography Mission has produced the most complete, high-resolution digital elevation model of the earth 2 grid, this information was used for computing average elevation and the coefficient of variation of elevation used as a measure of terrain ruggedness. Night light data was obtained from the United States Air Force Defense Meteorological Satellite Program (DMSP) and National Oceanic and Atmospheric Administration's (NOAA) Operational Linescan System (OLS) . Density of roads (length of paved road per km2), and Euclidean distances to roads, protected areas and night lights were computed in ArcGIS 9.3 (www.esri.com) software.Remotely sensed data that depict landscape characteristics and human impacts were obtained from various sources and extracted at the 10×10 km grid resolution. Forest cover was obtained from the Forest Survey of India 2) so as to ensure an even distribution of sampling effort across the landscape, analysis was done at the scale of 1,851 grids, each of size 10×10 km. This grid size was chosen since it was larger than the average home range size of a tiger Though sampling was done at the level of the forest beat (an administrative unit of about 15 kma priori expected to be positively influenced by (a) prey abundance, and (b) amount and quality of tiger habitat, and negatively influenced by (c) human disturbance http://www.proteus.co.nz/). Model selection was done using AIC, and model fit was assessed by comparing the actual detection histories with simulations generated from 50,000 parametric bootstrap runs of the target model in PRESENCE. The over dispersion parameter Ĉ close to one suggests that the model provides an adequate description of the data, values of Ĉ greater than one suggests more variation in the data than expected by the model, and values less than one suggest less variation in the data. The standard errors of model estimates were adjusted by the square root of Ĉ as recommended by MacKenzie et al.Ψ) in each grid.Tiger site occupancy was We used two approaches to estimate population extents and area of occupied habitats; (i) a more conservative approach wherein we considered only those grids that detected tiger sign as being occupied (the naïve estimate) and (ii) model inferred occupancy that corrected for detection bias and covariates in PRESENCE. Herein, landscape scale occupancy was computed by sum of cell occupancy probability values and divided by the total number of cells. Tiger habitat (forested area) in each grid was weighted by the tiger occupancy probability of that grid and summed across all grids to arrive at occupied tiger habitat for the landscape Ψ) obtained from PRESENCE was used as a measure of habitat suitability for tigers Ψ. For the non-tiger habitat (human land uses) where tiger occupancy values were not available, we considered them permeable to tigers at higher costs than forested habitats, although human habitations (townships) were considered impermeable to tigers  that result in long-term (FST) genetic differences between populations due to tiger movement across the landscape.The genetic structure between populations is a consequence of the amount of genetic drift to which each local population has been subjected, due to its local effective size, and/or due to its degree of demographic, geographic and ecological isolation FST, RST and PhiPT genetic distance estimates, obtained from AMOVA analysis in GENALEX, were linearized using the formula FST/(1 - FST), as given by Rousset 10 GGD), least-cost pathway distances (LCPD) least-cost corridor distances (LCCD), and resistance distances (RD). In addition, the effectiveness of the modeled corridors and spatial distance matrices was compared using partial correlations that allowed one model to be tested, while controlling for other competing models Pairwise As patterns of isolation-by-distance (IBD) are known to bias tests of hierarchical structuring and vice-versa, we used the population clusters as a covariate in a partial Mantel test, to model the partial correlations between pairwise genetic distances and spatial distances, while controlling the effect of population clusters (following Meirmans b (mtDNA cyt b) primers, of which 275 were identified as tiger scat based on BamHI restriction enzyme digestion profile of the PCR products . The total number of individuals identified here constitutes roughly 49% of the estimated total tiger population in the entire sampled area (PI-sib) of 1.5×10−4  of the samples was sufficiently low to determine unique individuals.Out of 587 scat samples, 330 scats were successfully PCR amplified using the felid specific mitochondrial DNA cytochrome products . From thled area . Sex ide1.5×10−4 , indicatp>0.05 at 1,000 permutations). Except for deviation at three loci, Fca441, Pati09 and Pati18 (p<0.001), all other loci were in Hardy-Weinberg equilibrium (HWE) in the pooled population after adjusting the critical P-value using the Bonferroni correction procedure (No significant evidence of linkage disequilibrium (LD) among all pair-wise loci combinations was observed, when all sampling locations were pooled . Partial correlations between estimated population size and the allele diversity indices, after controlling for the effect of sample size, were positive and highly significant . Heterozygosity values were not correlated with population size . Correlation of estimated population size with actual sampled size was highly significant , meaning that our samples were in proportion to the size of the population.Most tigers genotyped in this study showed high heterozygosity . The minpulation . A summapulation . The numpulation . EstimatPhiPT genetic distance estimator, individuals in the area were clustered into roughly four groups with varying degrees of population partitioning  based on itioning . The thrK from the output of the STRUCTURE runs using prior population information (locprior  = 1), produced the largest modal value of the statistic at K = 4, suggesting pronounced population subdivision at K = 4 (L(K) value reached an inflection point at K = 4 before gradually plateauing at K = 6 to 7 and finally leveling off at K = 8 (L(K) increased at higher values of K, as reported previously with other studies K and log-likelihood values occasionally occurs in cases where the FST values are significant Q summary barplots (K = 2 to 4 (carried out both with and without prior sampling location information), and distinct population saturation, indicative of population subdivision was evident at K = 4 in conformance with the delta K approach. Based on the above, the four cluster solution (K = 4) best describes the levels of genetic subdivision in our sample of the Central Indian tiger population  between the STRUCTURE identified clusters (p<0.01). Both FST and PhiPT values showed highly significant structuring (p≤0.001), and had relatively similar trends in magnitude with low sampling variance. In contrast, RST estimates showed no variation between groups, and had unreliably high sampling variances and mean square error estimates. The results of pair-wise FST and RST calculations indicated significant (p<0.05) and varied  genetic structuring between all sampled populations in Central India  to high (0.079 to 0.102), compared to pairwise estimates between different clusters (0.127 to 0.217).Partitioning of genetic variation in the AMOVA test indicated low but significant differentiation  and showed variable Q (0.289 to 0.758), and with the majority of samples having cluster memberships to more than one non-home locality.Eight putative migrants were identified in STRUCTURE . Of thesP<0.01), with high log likelihood of cross-assignment (h/LmaxL>2.0). Lowering the likelihood threshold (h/LmaxL<2.0) yielded further six putative migrants (P<0.05).GENECLASS also identified eight individuals  as putatP>0.8), and four individuals had intermediate migrant assignments (P = 0.509 to 0.617). In general, the posterior probabilities of migrant assignment were higher in BAYESASS compared to the STRUCTURE analysis. Additionally, BAYESASS identified six other individuals  as potential migrants (P = 0.509 to 0.926) that were not assigned as migrants by either STRUCTURE or GENECLASS.A total of fifteen individuals with likely immigrant or admixed ancestry were detected in the BAYESASS analysis . Of the Identification of offspring-candidate parent pairs in CERVUS yielded parentage relationships in thirteen out of seventeen putative migrants . No evidh/LmaxL>2.0 were classified as 100% first generation migrants. Two individuals  with>90% gen1 assignment and three individuals  with relatively high gen1 assignment probability (0.5 to 0.7) were considered as migrants. Five individuals  showed moderate levels of migrant assignment and immigrant ancestry patterns are indicative of admixed status. The assignment status of seven more individuals  was equivocal. While STRUCTURE could not assign them as migrants, they were identified as potential second generation migrants or admixed individuals in BAYESASS.The detection of migrants by the above methods yielded a total of seventeen individuals with putative immigrant ancestry . Identicm = 0.09) and Kanha to Pench (m = 0.07) where the rates were more than 5% (m = 0.015). Asymmetric migration was also visible between the Pench and Kanha population clusters, as the proportion of migrants from Pench to Kanha was negligible (m = 0.006). The Bandhavgarh population was devoid of migrants as suggested by the lack of gene flow between other populations (m<0.01). As in the locality-wise analysis, similar asymmetry and rates of migration were obtained between the various STRUCTURE defined population clusters (The mean posterior distributions of pairwise immigration rates depicting contemporary gene flow estimates in BAYESASS are shown in  than 5% . Gene flclusters .θ) and historical mutation scaled immigration rate (M) suggesting very low overall migrant proportions in the area over the long-term (M ranged from a high of 6.17 (Pench to Bandhavgarh) to a low of 0.9 (Bandhavgarh to Melghat-Satpura-Tadoba), revealing limited to no migration among populations in the landscape , to the population with the smaller theta value (Melghat-Satpura-Tadoba and Bandhavgarh clusters). The number of migrants per generation ranged from almost zero (Bandhavgarh to Melghat-Satpura-Tadoba cluster) to nine (Pench to Bandhavgarh). The Pench and Kanha clusters represented the largest source populations for immigrants in the area. Though marginally higher migration from Pench to Kanha was visible compared to migration in the other direction, both population clusters had overall symmetric gene flow.Results from the MIGRATE analysis showed low estimates of relative effective population size  regardless of the mutation models used and all localities tested showed normal L-shaped allele distributions in the mode-shift test, indicative of stable non-bottlenecked populations (p<0.05) evidence of heterozygosity deficiency  PC1 that represented availability of large undisturbed good canopy forests, (b) PC2 and PC4 that represented grids that had low human and livestock disturbances, (c) PC3, PC6 and PC9 that represented high ungulate prey especially in the form of chital, sambar, wild pig and gaur, (d) PC5 representing lower elevation, (e) PC7 representing grids within or in the proximity of legally protected areas and (f) PC8 that gave greater loadings for grids with higher rainfall , Ĉ statistic was estimated at 1.4 and standard errors shown in 2 area of the sampled grids, 76,913 km2 was forested or potential tiger habitat. The naïve estimate of grid occupancy was 17.5% while the model-averaged estimate of occupancy was 20.87% giving a 3.4% increment over the naïve estimate. The naïve estimate of tiger occupied habitat was 19,845 km2 while the model inferred occupancy of forests was 21,290 km2 giving an increment of 1.8% in occupancy estimate. The habitat variables of forest area, forest core area, rainfall, NDVI, and terrain ruggedness had significantly higher values in tiger occupied grids compared to unoccupied cells. All wild tiger prey indices were higher, while livestock abundance indices were lower in tiger occupied grids. Relevant human impact indices had significantly lower values in tiger occupied grids  had Eigen values greater than one and together explained 86.6% of the variability of the original variables . The facrainfall  and 8. Trainfall . The gooed grids .2 with intermittent tiger presence recorded throughout this patch even outside the legal reserve boundaries. The Satpura-Melghat forest patch was 12,720 km2, while the forest patches that contained Tadoba and Bandhavgarh Tiger reserves were smaller 2,088 km2 and 1,902 km2 respectively and connected to larger adjacent patches by fragmented forests  was recorded in Achanakmar  genetic distances vs. resistance  and least-cost corridor distances . In comparison to Euclidean geographic distance correlations, this corresponded to a 29.5% and 25.7% increase in model fit for the circuit theory based IBR and the LCC models respectively. The proportion of migrants was higher between population pairs that had lower corridor costs  and resistance distances . This meant a 46.2% and 31.6% increase over log-transformed Euclidean distance correlations for the circuit theory and corridor models respectively.Mantel's timators . The higor costs . LineariFST: r>0.48, p<0.02; linearized PhiPT: r>0.39, p<0.05). In contrast, after controlling the effect of resistance distance, significant positive correlation was only observed between linearized FST vs. geographic and least-cost corridor distances, while partial correlations between all other spatial and genetic distance metrics were either non-significant or negative. Although significant positive correlation was observed between FST and geographic distances in the standard Mantel tests, genetic and geographic distances were uncorrelated in the partial Mantel tests, after controlling the effect of population clusters, thus affirming that isolation-by-distance (IBD) pattern was absent in the data. Only resistance (IBR) and LCC distances retained a significant, positive partial correlation with genetic distances, when controlled for the effect of population clusters .Correlations in partial Mantel tests showed a significant, positive relationship between genetic and resistance distances, after controlling for all competing spatial distance metrics (linearized n = 169) representing about 49% of the estimated population The present study is illustrative of the general strengths and challenges in using non-invasive genetic samples to assess spatial distribution patterns in an endangered and cryptic species living in fragmented habitats. Although maternally inherited mitochondrial DNA could elucidate patterns of female gene flow, the sequencing of mitochondrial genes was not attempted as the primary objective of the study was to assess the functionality of corridors through the detection of first and second generation migrants. Hence, highly polymorphic, bi-parentally inherited, co-dominant and selectively neutral nuclear loci such as microsatellites were most suited for this purpose. Microsatellite marker amplification success was low from scats, reflecting the often poor quality of samples, typical of tropical environments Most individuals genotyped in this study, possessed heterozygous genotypes at>50% of typed loci, and there was no discernible differences in heterozygosity between migrant and resident individuals . HoweverHo = 0.701±0.059, He = 0.754±0.039) observed in our study is typical of genetic diversity prevalent among tigers in the Indian subcontinent He) and mean number of alleles (A) to be lower in other subspecies, viz. P. t. altaica , and allelic diversity (A = 7.76±1.96), and very low genetic subdivision (mean FST = 0.013±0.006). Another study in the southern parts of the Central Indian landscape, focused primarily in peripheral habitats just outside the scope of this study area Ho = 0.54, He = 0.81), and allelic diversity (A = 11.71) levels at fourteen microsatellite loci. A range-wide study Ho = 0.70±0.16) and allelic diversity (A = 12.4±3.6) compared to all other tiger subspecies . The study also reported low and non-significant genetic structuring of the Central Indian tigers with the Northern  and Southern Indian populations . This could be attributed to a historically large effective population size and inter-population connectivity in the region of Central and Peninsular India, explaining why, despite centuries of immense trophy hunting and continued habitat fragmentation, extant tiger populations in the region currently retain close to 60% of the global genetic variation in the species The allelic diversity (9.1±2.2) and heterozygosity  resulting in higher hierarchical levels of population subdivision at K = 8. In contrast, the runs carried out with population information  did not detect unnecessary genetic structuring, and ignored the prior sampling location information if the ancestry of individuals was uncorrelated K = 4 appeared optimal for the following reasons. In cases where STRUCTURE detects multiple clustering options with similar probabilities, typically the lowest K value which captures much of the biological complexity in the sample is considered to be the most conservative K value FST statistics indicated significant pairwise structuring across all populations in the area, the pattern shared certain similarities to the population clusters identified by STRUCTURE and the ordination results in PCoA. FST values were generally lower for localities within the same cluster compared to pairwise estimates between different clusters. We therefore treated the genetic distance estimators including FST and its analogues as relative measures of population differentiation. The estimation of these parameters requires prior identification of populations and unless the population units are clearly known, such a priori designation may not reflect realistic biological patterns as they would only be representing ad hoc division of populations FST and its analogues are based may not be suited for estimating genetic differences between populations that have undergone fragmentation or demographic fluctuation events only recently. On the other hand, the results of the model based STRUCTURE clustering approach, which partitioned individuals into relatively distinct clusters based on iterative assignments, made much more sense of the biological realities of the area as the structuring observed here is likely an artifact of recent population fragmentation.By using a combination of classical population differentiation and individual clustering approaches, we were able to detect patterns of population sub-structuring in the region. Results from both the PCoA and STRUCTURE analyses suggest the presence of four genetic clusters in the area. The clustered localities occur in close proximity to each other and the overall pattern of genetic structuring observed in the landscape is concordant with existing habitat connectivity and indicative of the role that habitat fragmentation plays in shaping the distribution of allele frequencies across populations. Significant genetic structuring was also detected in the population pairwise FST and RST values can provide valuable insights into the balance between genetic drift and mutation events in the studied populations FST values compared to RST values. The FST statistic is based on allelic identity and accounts for gene flow between different populations as the basic premise under which it estimates pairwise genetic differences RST relies on allele size and single stepwise mutations are the primary contributors of genetic variation for this statistic RST estimations produced non-significant results across many pairwise comparisons, and had higher sampling variances compared to FST. This suggests that populations in the area were not isolated long enough for mutations to have caused the genetic differences between populations and the primary cause of genetic structuring in the area is due to recent genetic drift. Some measure of this drift is evident from the number of private alleles found in each population, especially Bandhavgarh, which has the highest individual allele frequency compared to all other localities .Although the STRUCTURE and PCoA results suggest subdivision at four clusters for the Central Indian tiger population, the significant 2) to low (Achanakmar 0.1 tigers/100 km2) are juxtaposed in a matrix encompassing a range of land-use regimes from undisturbed natural forest to high density human settlements . In these situations it was difficult to tease apart actual migration events from similar allelic patterns that arose from shared population histories and low genetic separation. On the other hand, migrant identification between localities in different population clusters produced robust assignments because of distinct genetic differences. Migrant detection was highest between localities that had discernible genetic separation and had relatively intact habitat corridors with confirmed tiger presence, such as between Kanha and Pench populations where four migrants and seven likely admixed individuals were detected. Other studies by Joshi Q>0.8) to a single non-home cluster, as first generation migrants. Further, a fourth male individual (D1853) that had high cross-assignments, albeit with marginally lower Q value (0.527) compared to the above four migrants, was also designated as a first generation migrant. The immigrant ancestry status in the remaining ten putative migrant individuals was classified as admixed based on - (i) intermediate levels of migrant probability in STRUCTURE; (ii) membership to more than one cluster in the individual Q assignments; and (iii) high probability of assignment to second and not to first generation immigrant ancestry state in STRUCTURE and BAYESASS.Based on our strict criteria of defining migrants, we designated four individuals, three males  and one female (D958) that had high cross-assignments , confirming that dispersal is male-biased in tigers et al.et al.et al.2) likely serving as a source population, while the population density in Melghat is lower (2 tigers/100 km2). Asymmetric contemporary rates of gene flow were also apparent between Pench and Kanha, as migration from Kanha (6 tigers/100 km2) to Pench was much higher compared to movement in the opposite direction from Pench to Kanha. Joshi et al.et al.et al.Both the contemporary and historical analyses detected low estimates of migration rate between populations in the landscape. While this result is superficially similar to the findings of Joshi Contemporary density estimation studies using photographic capture-recapture techniques in the region Ne past generations, As parameter estimates of migration rate and theta in MIGRATE average across at least 4et al.M ratios (<0.68) suggestive of bottleneck, but the evidence was equivocal since no significant heterozygosity excess or a mode-shift in allele frequencies was detected. Though bottlenecks were not observed in this study, the analyses may be undermined in a few populations because of low sample sizes , as tests such as the mode-shift in allele frequencies is known to be affected by sample size variances et al.Alternatively, the low estimates of historic migration rate raise concerns whether these results are artifacts of sampling or population related. MIGRATE parameter estimations assume coalescent-based models of constant population sizes and mutation-migration-drift equilibrium. Departures from the equilibrium model such as recent and sudden declines in population sizes can negatively bias the posterior parameter distributions of theta and hence migration rate estimations et al.et al.et al.et al.et al.Our study and the recent study by Joshi 2 of tiger habitat. Thus a 10×10 km2 grid had anywhere between 3 to 35 replicate surveys depending on the amount of tiger habitat present within that grid. The detection probability of tiger sign was high in comparison to other studies Occupancy has often been used as a straightforward and economical state variable in place of abundance to monitor populations a priori predictions based on our understanding of tiger ecology. Since PCs are orthogonal to each other a priori predicted effects and to the coefficients obtained by the multivariate best model obtained from their PCs by PRESENCE. These results indicate that tigers are a conservation-dependent species, requiring areas having effective legal protection, good forest cover with least human impacts and high prey availability.Detection of tiger sign was better modeled by including encounter rate of sign per km walk. Intensity of sign was found to be a reliable index of tiger abundance Although both LCP and circuit theory used tiger occupancy probability as a base layer to parameterize landscape resistance surfaces, LCP defines the optimal minimal route that is required to connect two tiger reserves FST). Instead, the LCCs which were delineated by matching and buffering the pathway results from the raw LCP model with existing ground forest cover maps of the area provided a more realistic relationship of the ecological costs with observed genetic differences. In contrast, Euclidean distances (GGD and log10GGD) did not correlate significantly with genetic distances, due to the null model assumptions of spatial homogeneity where habitats are arrayed in an infinite lattice FST (but not PhiPT or RST) with geographic distance is an artifact of metapopulation structure and discrete population clusters present in the area. This observation was affirmed by non-significant partial correlations between genetic vs. geographic distances (but not LCCD and RD) using the STRUCTURE identified population clusters as a control. Apart from fragmentation induced spatial heterogeneity, genetic structure is also a result of population history.In our study, the ecological distances that were generated using information from habitat heterogeneity and landscape resistance surfaces (LCCD and RD), correlated significantly with pairwise genetic distances, which validated the effectiveness of the modeled linkages in representing realistic biological scenarios. In particular, IBR models depicted well the ecological costs of movement as they accounted for multiple pathways, irregular patch effects, landscape heterogeneity and wider habitat swaths connecting populations The results of our study imply that population subdivision and genetic structure across most localities in the area was strongly associated with habitat features that offer resistance to dispersal at different intensities such as agricultural land, roads, railway lines, high density human settlements and urban infrastructure and not only by geographic distance between populations. Mantel tests showed significant positive correlation of genetic differentiation with least-cost corridors and landscape resistance surfaces associated in traversing corridors, further confirming this observation. The tiger habitats in the region are patchy with some populations still having connectivity, while being reduced or conspicuously absent in others. The best patches of contiguous forested habitat are present in the corridor between Pench and Kanha, which extends eastward to Achanakmar. Likewise, the Satpura and Melghat populations are also connected through swathes of degraded forests, which are interspersed with agricultural land and medium density human settlements. Connectivity between Pench and Satpura is fragile as parts of the linkage are disrupted by mining activities, and broken up in places by agriculture, habitations, major highways and railway lines. The population in Tadoba is linked with Kanha in a stepping stone connectivity through patchily distributed forests. Even though the intervening matrix between Tadoba, Pench and Melghat is heavily human dominated, the populations are tenuously linked by degraded forest patches and tiger occupied habitats such as Bor Wildlife Sanctuary. Bandhavgarh has linkages with forest habitat and tiger reserves further east It is indeed surprising that in spite of being highly fragmented Elephas maximus), gaurs (Bos gaurus), leopards (Panthera pardus), and dholes . They need to be offered legal protection with smart green infrastructure Conservation efforts in such a fragmented and dynamic human-dominated landscape presents enormous challenges, but should be attempted in all areas where substantial habitat is still present. Our study gives renewed hope to tiger conservation efforts within the Central Indian Landscape and similar habitats with small fragmented tiger populations. We show that minimal habitat connectivity permits gene flow between populations, which is essential to maintain metapopulation connectivity. Our findings suggest that tigers could negotiate passage through stepping stone dispersal Figure S1A) MtDNA cyt b alignment with GenBank and reference sequences showing polymorphism at the particular BamHI restriction enzyme between tiger and leopard. (B) Enzyme digested bands of the 187 bp PCR product, targeting this region, showing different profiles in tiger and leopard for species identification.PCR-RFLP identification of tiger scats. ((TIF)Click here for additional data file.Figure S2Frequency of heterozygous genotypes observed at each individual multilocus genotype in all tiger individuals (n = 169) in this study.(TIF)Click here for additional data file.Figure S3A). Difference in delta K and mean LnP(K) for an estimated number of K populations, in models run with (locprior  = 1) and without (locprior  = 0) prior sampling location information. (B). Summary barplots depicting prior and non-prior STRUCTURE runs (assumed K = 2 to 8), of sampled populations in central India showing cluster affiliations according to individual Q values. Cluster saturation at K = 4, indicative of four population clusters, is observed in runs carried out both with and without a priori location information. At K>4, increased sub-structuring is detected, but there is no concordance in clustering between the prior and non-prior runs.Results of STRUCTURE analysis. ((TIF)Click here for additional data file.Figure S4Differences in tiger present (n = 311) and tiger absent (n = 1540), 10×10 km grids shown as violin plots in the Central Indian Landscape. All variables are normalized by z transformation to make the scales comparable.(PDF)Click here for additional data file.Table S1Ho) and expected (He) heterozygosity, Hardy-Weinberg equilibrium (HWE) tests, null allele frequencies and sibling probability of identity (PI-sib) values obtained across 169 tiger individuals. Null allele frequencies>+0.05 are italicized.Information on the 11 microsatellite loci used in this study. Allele diversity statistics, observed ((DOCX)Click here for additional data file.Table S2k), number of individuals typed (N), observed (Ho) and expected (He) heterozygosity, Hardy-Weinberg equilibrium (HWE) test significance and null allele frequencies (Null). The Achanakmar (n = 4) and Kanha-Pench corridor (n = 5) individuals are included in the Kanha cluster.Summary of population cluster-wise genetic diversity statistics at each locus. Depicted are number of alleles ((DOCX)Click here for additional data file.Table S3List of diagnostic alleles present in the sampled populations.(DOCX)Click here for additional data file.Table S4AMOVA results.(DOCX)Click here for additional data file.Table S5FST  and RST  estimates.Population pair-wise (DOCX)Click here for additional data file.Table S6P values <0.05 are depicted in italics.Bottleneck test results for loci under different mutation models. (DOCX)Click here for additional data file.Table S7Variable attributes used as site covariates in modeling tiger occupancy.(DOCX)Click here for additional data file.Table S8Habitat corridors, major roads, corridor cost between tiger reserves.(DOCX)Click here for additional data file.Table S9r) and probability (p) are shown using three different genetic distance estimators. Significant values (p<0.05) are indicated by an asterisk (*).Results of standard and partial Mantel tests for correlation between pairwise genetic and spatial distance metrics. The correlation coefficient ((DOCX)Click here for additional data file.Table S10b PCR amplification in reference samples using felid-specific (187 bp) and universal primers (309 bp). + indicates all samples which amplified. NA – not amplified.Details of cyt (DOCX)Click here for additional data file.Table S11n = 65) for species identification of tiger samples by PCR and BamHI restriction enzyme digestion.Information on the pilot test carried out on scats ((DOCX)Click here for additional data file.Method S1Species identification from scat samples.(DOCX)Click here for additional data file.Method S2Generating the cost surface for PATHMATRIX and CIRCUITSCAPE.(DOCX)Click here for additional data file.


Use of mechanically-assisted spinal manipulation (MA-SM) devices is increasing among manual therapy clinicians worldwide. The purpose of this pilot study is to determine the feasibility of recording 5-6 and L6-7 facet joints of a cat spine. Three L6muscle spindle afferents with receptive fields in back muscles were isolated. Recordings were made during MA-SM thrusts delivered to the L7 spinous process using an instrumented Activator IV clinical device.Intervertebral fixation was created by inserting facet screws through the left LNine MA-SM thrusts were delivered with peak forces ranging from 68-122N and with thrust durations of less than 5ms. High frequency muscle spindle discharge occurred during MA-SM. Following the MA-SM, muscle spindle responses included returning to pre-manipulation levels, slightly decreasing for a short window of time, and greatly decreasing for more than 40s.in vivo muscle spindle response using clinical MA-SM devices in an animal model is feasible. Extremely short duration MA-SM thrusts (<5ms) can have an immediate and/or a prolonged (> 40s) effect on muscle spindle discharge. Greater peak forces during MA-SM thrusts may not necessarily yield greater muscle spindle responses. Determining peripheral response during and following spinal manipulation may be an important step in optimizing its’ clinical efficacy. Future studies may investigate the effect of thrust dosage and magnitude.This study demonstrates that recording  Spinal manipulation is a form of manual therapy commonly used by clinicians and therapists for conservative treatment of musculoskeletal complaints. Spinal manipulation is typically distinguished from spinal mobilization by the presence of a short duration mechanical thrust applied to the spinal column using either direct hand contact ≤150ms) or one of several commercially available mechanical devices ≤10ms) 50ms or o. Among cms 50ms oSpinal manipulation has been shown to be effective in the treatment of neck and low back pain and is recommended by clinical guidelines and evidence reports -16. Sevein vivo animal [in vivo feline model of intervertebral joint fixation.Spinal manipulation by its very nature is a mechanical stimulus typically applied at clinically identified sites of intervertebral joint fixation or joint hypomobility. Theorized mechanisms for its therapeutic effects include breaking of joint adhesions and/or alteration of sensory input from primary afferents of paraspinal tissues which subsequently act to influence spinal cord reflexes and/or other central neural mechanisms ,33. MA-So animal ,36 and ho animal ,23,29 sto animal . Muscle o animal -40. Howe6 spinal manipulation delivered by a computer controlled feedback motor.The experimental preparation and procedures used in this study have been described in greater detail elsewhere -42 and a5 laminectomy was performed exposing the right L6 dorsal rootlets which were cut close to their entrance to the spinal cord and placed on a platform. Thin filaments were teased with fine forceps until action potentials from a single neuron were identified that responded to both mechanical pressure applied directly to the paraspinal back muscles (multifidus or longissimus) and a fast vibratory stimulus . Afferent fibers remained positioned on the recording electrode while facet screws  were inserted through the left articular pillars of L5-6 and L6-7 vertebra in similar fashion to that previously described [5-6 and L6-7 facet fixation is shown in Catheters were placed in the common carotid artery and external jugular vein to monitor blood pressure, introduce fluids and/or supplemental anesthesia if the arterial pressure rose above 120mm Hg or if a withdrawal reflex became present. The trachea was intubated and the cat was artificially ventilated. Since our focus was on back afferents, the right sciatic nerve was cut to reduce afferent input from the hindlimb. An Lescribed . An x-raThe Activator IV  is a hand-held clinical device comprised of a rubber-tipped spring-loaded hammer with 4 device settings that produce relative increases in thrust magnitude. Its thrust duration is<10ms and can deliver a maximum force of 212N when tested directly on a load cell [7 spinous process  and a small preload was applied. The L7vertebra was chosen to receive the MA-SM thrust due to the increased risk of tearing the L6 afferent fiber off the recording electrode during an L6 manipulation. The Activator IV device requires that a preload force be applied in order to completely retract the instrument tip prior to triggering the manipulative thrust. We used the two lowest device settings (1 and 2) which can deliver a force of 123N when tested directly on a load cell [Once a single back afferent had been isolated, the Activator IV device was placed by hand directly onto the exposed fascia overlying the cat’s Lsettings  and 2 wh7 MA-SMs in a single cat preparation with intervertebral joint fixation. All afferents responded to mechanical movement of the spine and had sustained responses to fast vibratory stimuli (~70 Hz). All 3 afferents received MA-SM thrusts at a device setting of 1, whereas 2 afferents also received MA-SM thrusts at a device setting of 2. Individual MA-SM thrust profiles are reported in Three muscle spindle afferents with receptive fields located in the longissimus back muscle were recorded during 9 LExamples of spindle responses to MA-SM thrusts from Afferent 1 and 2 are shown in 7 spinous process . Evidence for a lack of damage to the afferent fiber is in part provided through the return of pre-thrust resting spindle discharge following MA-SM. The risk of potential afferent fiber damage during MA-SM delivery in this preparation is real, but can be minimized by using dorsal rootlets that are longer in length. Although noise artifacts were encountered during the experiments, this appeared due in large part to movement of the device while the operator delivered the thrust. This issue can be remedied by non-manually triggering the MA-SM device attached to a rigid frame or perhaps using newer electrically powered (non-spring-loaded) MA-SM devices [Although this pilot study contained a limited number of afferents, it demonstrated some important findings and will help to inform future studies. First with regards to the preparation, we demonstrated the feasibility of recording muscle spindle responses in an  devices .7 spinous process thrust site, and/or other biological factors. In a previous study investigating the effects of L6 and L7 anatomical thrust delivery sites on L6 muscle spindle discharge, we found that segmental contact sites distant to the muscle spindle’s receptive field were just as effective at increasing spindle discharge as contact sites close to the receptive field [We found that the extremely short MA-SM thrust durations elicited high frequency discharge from paraspinal muscle spindle afferents. This response appears similar that which occurs during 25-150ms thrust durations delivered by a computer-controlled feedback motor -40 Figu, but dirve field .3 produced larger adjacent vertebral motions at shorter thrust durations (10ms) compared to longer thrust durations (100 and 200ms) [We found it interesting that the lower force delivered at setting 2 (78.2N) versus the higher peak force delivered at setting 1 force (107.9N) had a greater impact on Afferent 3’s discharge post-thrust . It is rd 200ms) ,31. It id 200ms) . HoweverPreload forces and preload durations were not standardized in the current study as the Activator IV device was operated by hand as is performed clinically. Applied preload forces are required to retract the tip of Activator IV device, but we consciously attempted to limit the magnitude of applied preload forces since the preload duration was not standardized. We used thrust force magnitudes in our animal model that were the same or similar to those used in human studies in the human cervical spine. In humans, mean peak forces during manually applied cervical manipulation has been reported to be 118N . Althougin vivo muscle spindle response during spinal manipulation using clinical mechanically-assisted spinal manipulation devices. It also demonstrates that extremely short duration manipulative thrusts (<5ms) of equivalent forces to that delivered to the human cervical spine can have an immediate and/or perhaps a prolonged effect (> 40s) on paraspinal muscle spindle discharge. While the clinical relevance of how mechanoreceptor stimulation or inhibition related to spinal manipulation modulates central nervous system activity remains to be clarified, determining how various mechanoreceptors respond during and following spinal manipulative thrusts in a clinically relevant fashion is an important step toward achieving this goal.This pilot study demonstrates feasibility of recording


We have previously shown that hyperuricemia plays an important role in the vascular complications of insulin resistance (IR). Here we investigated the effect of xanthine oxidase (XO) inhibition on the cardiac complications of IR.1) were assessed by immunofluorescence.IR was induced in rats by a high fructose high fat diet for 12 weeks. Allopurinol, a standard XO inhibitor, was administered in the last 4 weeks before cardiac hemodynamics and electrocardiography, serum glucose, insulin, tumor necrosis factor alpha (TNFα), 8-isoprostane, uric acid, lactate dehydrogenase (LDH) and XO activity were measured. Expression of cardiac angiotensin II (AngII) and angiotensin receptor 1  and XO inhibition greatly attenuated impaired relaxation. IR was accompanied by cardiac ischemia  while XO inhibition alleviated the ECG abnormalities. When subjected to isoproterenol-induced ischemia, IR hearts were less resistant (reflected by larger ST height depression and higher LDH level) while XO inhibition alleviated the accompanying ischemia. In addition, XO inhibition prevented the elevation of serum 8-isoprostane and TNFα, and blocked elevated AngII and ATXO inhibition alleviates cardiac ischemia and impaired relaxation in IR through the inhibition of low grade inflammation and the angiotensin system. Insulin resistance (IR) is a key component of metabolic syndrome (MetS). The pathogenesis of the syndrome includes multiple organs involved in the cardiovascular system . The prein vitro incubation of normal isolated aorta with uric acid at the same concentration observed in IR animals impairs endothelial dependent relaxation [Although hyperuricemia is condition that frequently accompanies MetS, its role as a risk factor for cardiovascular complications in MetS is debated. For decades, hyperuricemia was considered as a simple result of renal dysfunction and not as a true mediator but in the late 1990s researchers started to reevaluate hyperuricemia as a causal risk factor for cardiovascular disease . We havelaxation .In addition to uric acid production, the xanthine oxidase (XO) enzyme generates reactive oxygen species (ROS) through catalysing oxidation of hypoxanthine to xanthine and then to uric acid. XO-derived reactive oxygen species (ROS) contribute to many pathologic conditions characterized by ischemia or inflammation . AllopurTherefore, the aim of the present work was to investigate whether XO represents a realistic target for intervention in the context of protection of cardiovascular dysfunction in IR and to suggest the possible mechanism of action for protection. Data presented from this animal study indicate potential new therapeutic strategies for the treatment of human disease.The study is reported in accordance with the Animals in Research: Reporting In Vivo Experiments (ARRIVE) guidelines for research involving animals , and the−1.day−1) was administered daily in the last 4 weeks by dissolving in drinking water (100–110 mg.l−1) depending on daily water consumption [The experimental protocol was approved by the Unit of Biomedical Ethics Research Committee, King Abdulaziz University, KSA. Animals were randomly divided into three groups  of control (C), IR or allopurinol-treated insulin resistance (IR-Allo). IR was induced by adding fructose 10%) to drinking water and feeding rats on high fat high salt diet  for 12 weeks. Control animals received water and standard diet . Allopurinol  injection [−1) injection through the femoral vein [1), collagen I and 4-hydroxy-2-noneal Michael adducts (4HNE) in heart cross sections.At the end of the study animals were fasted for 8 hours. A tail capillary droplet was used to determine the fasting blood glucose level before rats were anaesthetized by urethane . Serum insulin level was measured by enzyme-linked immunosorbent assay  using plates coated with anti-rat insulin antibodies. Serum TNFα and adiponectin levels were determined by ELISA using Quantikine® kits  using rat TNFα or rat adiponectin and antibodies raised against rat TNF-α or rat adiponectin respectively. XO activity was measured in the final serum sample using the BioVision® assay kit . Serum level of 8-isoprostane was determined by a competitive immunoassay 8-isoprostane EIA® kit . Serum levels of uric acid and triglycerides and LDH activity were determined using the ELITech® assay kit . Serum level of fructosamine was determined using Quimica Clinica® assay kit .Invasive real time recording of cardiac hemodynamics was carried out according to the method described in a previous report from our laboratories . FollowiThe standard surface ECG was recorded by the Powerlab® system  connected to a PC running LabChart professional software with the ECG module to detect different components of the ECG. The change in the ST segment height was used as the index of anginal severity and the ST segment value was defined as the mean ECG voltage 12 ms after the S wave peak , and the1, 4HNE and collagen protein in rat paraffin embedded heart sections (5 μm) was carried out according to the method used in our previous work [1 receptor , mouse monoclonal anti collagen type I , rabbit polyclonal anti 4HNE . The secondary antibodies used were Alexa Fluor (λex = 488) conjugated goat anti-mouse and Alexa Fluor (λex = 594) conjugated goat anti-rabbit .Immunofluorescence staining of AngII, ATous work -17. FixeThe following drugs and chemicals were used: Allopurinol, urethane . Allopurinol was dissolved in distilled water while urethane was dissolved in saline.Values are expressed as mean ± SEM. Statistical analysis was carried out by analysis of variance (ANOVA) followed by Newman-Keuls’ post hoc test using statistical software .High fructose high fat diet administration was associated by a significant elevation in serum uric acid level compared to control  development after twelve weeks feeding on high fructose and high fat diet was evidenced by the significantly higher weight gain p < 0.01), glucose (p < 0.001), insulin (p < 0.05) and triglyceride (p < 0.05) levels in the IR group than those in the control group. XO enzyme inhibition by allopurinol administration in the last four weeks did not significantly affect the exaggerated weight gain, the developed hyperglycemia, hyperinsulinemia or hypertriglyceridemia associated with the high fructose and high fat diet  XO inhibition ameliorated the impaired left ventricular diastolic relaxation associated with IR; (ii) the prolongation of cardiac repolarization in IR was alleviated by XO inhibition; (iii) XO inhibition alleviated the exaggerated cardiac ischemia following isoproterenol injection in IR; (iv) XO inhibition alleviated the low grade inflammation and oxidative stress associated with IR; (v) XO inhibition blocked the IR-induced activation of the angiotensin system both in heart tissue and coronary artery. These findings provide convincing evidence that XO inhibition offsets the cardiac diastolic dysfunction and ischemia accompanying IR in addition to the associated low grade inflammation and oxidative stress.The predominant feature in cardiomyopathy associated with diabetes, metabolic syndrome and IR seems to be the left ventricular diastolic dysfunction as shown in the present study and indicated in previous works ,19. Vent+ channels [Prolongation of cardiac repolarization is an important cardiovascular risk factor in several diseases, including diabetes mellitus and infarction . Animalschannels , and/or channels .The results of the present study show that animals with IR are less resistant to isoproterenol-induced acute cardiac ischemia as indicated by the significantly larger depression in ST height and higher serum LDH activity compared with control. This is in agreement with clinical literature where MetS is associated with a high incidence of coronary atheroma and an increased risk of fatal coronary events . The fruThe protective effect of XO inhibition against cardiac diastolic dysfunction and ischemia in animals with IR seems to be direct cardiovascular protection rather than affecting IR itself, because XO inhibition did not have a significant effect on the IR index. However, it did significantly alleviate the cardiac diastolic dysfunction and ischemia in animals with IR. This also provides proof for the assumption that hyperuricemia and XO activation are independent risk factors for cardiovascular disease in IR. Numerous current epidemiological studies point to a possible role of uric acid as an independent risk factor for cardiovascular complications in metabolic syndrome, but without definitive conclusions .in vitro incubation with uric acid at the concentration observed in MetS animals impaired endothelial relaxation in isolated normal aorta [The mechanism(s) by which XO inhibition alleviates cardiac diastolic dysfunction and ischemia in animals with IR could be by a reduction of the associated hyperuricemia and oxidative stress. Although uric acid has antioxidant effects extracellularly, it stimulates inflammatory and oxidative stress mechanisms inside the cell . This real aorta . In the Oxidative stress and low-grade inflammation are clearly involved in pathogenesis of the IR and MetS and its complications . The rea1 or AT2 receptors to exert different biological functions. Binding of AngII to AT1 has been shown to produce the second messengers inositol trisphosphate and diacylglycerol which lead to stimulation of reactive oxygen species and vasoconstriction in numerous previous reports . In the in mice .The possibility that modulation in serum lipids contributed to IR-XO cardiac dysfunction was also examined. Dyslipidemia is associated with increased cardiovascular mortality and morbidity . Our finXO inhibition by allopurinol alleviates cardiac diastolic dysfunction and ischemia associated with IR. Inhibition of hyperuricemia, low grade inflammation and the angiotensin system are suggested potential mechanisms for the observed cardiac protection. It remains to be determined whether the cardiac dysfunction observed is the consequence of one cytokine or of the combinatorial effects of multiple mediators, and the effect of blocking individual cytokines in this model may provide further mechanistic insight. The findings from this study suggest that xanthine oxidase may provide a novel drug target for the treatment of cardiac complications associated with insulin resistance and diabetes.