Is paternal obesity associated with epigenetic marks and weight status in offspring?This cohort study of 429 father-mother-infant triads found that paternal body mass index at the time of conception was associated with both offspring birth weight and epigenome-wide DNA methylation patterns in offspring at birth, age 3 years, and age 7 years.These findings suggest that paternal obesity may be an underappreciated contributor to childhood health outcomes. This cohort study investigates the associations between periconception paternal body mass index (BMI) and DNA methylation patterns in offspring. While prenatal nutrition and maternal obesity are recognized as important contributors to epigenetic changes and childhood obesity, the role of paternal obesity in the epigenome of offspring has not been well studied.To test whether periconception paternal body mass index (BMI) is associated with DNA methylation patterns in newborns, to examine associations between maternal and paternal BMI and the epigenome of offspring, and to examine persistence of epigenetic marks at ages 3 and 7 years.Project Viva is a prebirth cohort study of mothers and children including 2128 live births that enrolled mothers from April 1999 to July 2002 and followed offspring to adolescence. This study analyzed the subset of participants with available data on paternal BMI and DNA methylation in offspring blood in the newborn period, at age 3 years, and at age 7 years. Data were analyzed from July 2017 to October 2019.The primary exposure was paternal periconception BMI; associations were adjusted for maternal prepregnancy BMI and stratified according to maternal BMI above or below 25.The primary outcome was genome-wide DNA methylation patterns in offspring blood collected at birth, age 3 years, and age 7 years.z score, 0.38 [0.91] vs 0.11 [0.96]; P = .004). Cord blood DNA methylation at 9 CpG sites was associated with paternal BMI independent of maternal BMI (q < .05). Methylation at cg04763273, between TFAP2C and BMP7, decreased by 5% in cord blood with every 1-unit increase in paternal BMI (P = 3.13 × 10−8); hypomethylation at this site persisted at ages 3 years and 7 years. Paternal BMI was associated with methylation at cg01029450 in the promoter region of the ARFGAP3 gene; methylation at this site was also associated with lower infant birth weight  and with higher BMI z score at age 3 years.A total of 429 father-mother-infant triads were included. The mean (SD) periconception paternal BMI was 26.4 (4.0) and mean maternal prepregnancy BMI was 24.5 (5.2); 268 fathers had BMI greater than or equal to 25  and 161 had BMI less than 25 . Paternal BMI greater than or equal to 25 was associated with increased offspring birth weight compared with paternal BMI less than 25 (mean [SD] z score in offspring.In this study, paternal BMI was associated with DNA methylation, birth weight, and childhood BMI  Our group and others6 have reported that paternal undernutrition in rodents is associated with increased risk of obesity, diabetes, and hepatic steatosis in offspring. Additional paternal exposures, including low-protein diet, high-fat diet, hyperglycemia, psychological stress, and endocrine-disrupting chemicals, are also associated with behavioral responses, obesity, and diabetes risk in progeny in animal models.13 While epigenetic factors in male germ cells are thought to contribute, the precise mechanisms for nongenetic, paternal inheritance remain incompletely defined.Nutritional and environmental exposures during early development are associated with long-term health and disease.14 Obesity-associated epigenetic changes in sperm have been reported at imprinted genes15 and at genomic loci linked to appetite and weight regulation.14 Whether these obesity-associated epigenetic changes in germ cells might persist or alter early development in offspring is still unclear. Intriguingly, interindividual variation in body mass index  in humans was recently reported to be associated with methylation at a putative metastable epiallele in the pro-opiomelanocortin (POMC) gene, and analysis of family trios indicated that paternal somatic DNA methylation correlated strongly with DNA methylation patterns in offspring, whereas maternal methylation did not.16 Moreover, in the Newborn Epigenetics Study (NEST) cohort, paternal BMI greater than 30 was associated with reduced DNA methylation at several imprinted loci in umbilical cord leukocytes,17 but maternal vs paternal obesity had differential associations with DNA methylation of IGF2.18 However, few studies have examined the effects of paternal obesity on offspring epigenetic marks on a genome-wide basis, or the degree to which such marks may be associated with childhood weight status.Recent clinical studies have shown that obesity and weight loss may alter DNA methylation and expression of noncoding RNA in sperm, raising the possibility that paternally transmitted epigenetic inheritance could occur in human populations.We investigated the independent associations of paternal and maternal BMI with the offspring epigenome with the goal of identifying individual genomic loci and genomic features associated with paternal obesity. For those genomic regions that were differentially methylated in newborns relative to paternal BMI, we examined the extent to which DNA methylation patterns were retained in later life and associated with childhood weight status. We report that paternal periconceptional BMI is significantly associated with cord blood DNA methylation in newborns. For some loci, this association persists in early childhood leukocytes at ages 3 years and 7 years, highlighting an important association of paternal obesity with the childhood epigenome and metabolic risk.19 Briefly, pregnant women were recruited during their first prenatal visit from obstetric offices of Atrius Harvard Vanguard Medical Associates in eastern Massachusetts between April 1999 and July 2002; the current analyses were done between July 2017 and October 2019. Maternal prepregnancy BMI was calculated from self-reported height and prepregnancy weight, assessed at the first prenatal visit (median 9.9 weeks’ gestation). At each visit, research assistants measured weight with a digital scale and child’s height using a calibrated stadiometer. We used data from the Centers for Disease Control and Prevention 2000 growth charts20 to calculate weight-for-age z scores and BMI percentiles.We analyzed participants in Project Viva, a US longitudinal prospective birth cohort study of mothers and children. Detailed protocols, study design, and methods used for Project Viva have been previously published.Assessment of paternal BMI was based on maternal report of paternal weight and height at study enrollment. Of the total 2128 mother-infant pairs in the cohort, cord blood DNA methylation data were available for 485 infants, and BMI was available for 470 fathers. We excluded infants with gestational age less than 34 weeks and infants born to mothers with gestational, type 1, or type 2 diabetes or preeclampsia. Thus, a total of 429 father-mother-infant triads were available for analysis. Of these, 107 had blood methylation data available in early childhood  and 400 had blood methylation data available during midchildhood . Cord blood samples were collected between 1999 and 2003, early childhood samples between 2003 and 2006, and midchildhood samples between 2007 and 2010.STROBE) reporting guideline.We evaluated persistence of cord blood associations in children with available blood methylation measurements in early childhood and midchildhood with complete exposure and covariate information. Participant flow is shown in eTable 1 in the Trained medical personnel obtained umbilical cord blood at delivery; additional nonfasted blood samples were obtained during early childhood and midchildhood study visits. Cord blood samples were stored in a dedicated refrigerator at 4 °C and transported to a central location within 24 hours of sample collection. Similarly, whole blood samples collected during early childhood and midchildhood were stored at 4 °C and transported to the central storage location for sample processing; trained laboratory staff processed the samples on the day of arrival. Whole blood samples were centrifuged to separate the buffy coat from plasma and red blood cells, and the buffy coat was transferred into a red blood cell lysis solution. The solution was then centrifuged to obtain the white blood cell pellet. Aliquots were stored at −80 °C until analysis.Whole blood DNA was extracted using the Puregene Kit (Qiagen) and bisulfite converted using the EZ DNA Methylation-Gold Kit (Zymo Research). Samples were randomly allocated to chips and plates and analyzed using Infinium Human Methylation 450 BeadChip (Illumina Inc), which interrogates more than 485 000 CpG sites simultaneously at a single-nucleotide resolution, covering 99% of RefSeq genes; assays were performed in 2014.P < .05 after correcting for multiple comparisons using false discovery rates and Bonferroni correction. We adjusted for maternal BMI in our primary analyses (model 1). In parallel, to test whether the association between paternal BMI and offspring epigenetic marks may be influenced by maternal obesity, we also analyzed the association between paternal BMI and offspring epigenetic marks stratified according to maternal BMI categories . We additionally adjusted for maternal age, gestational weight gain, household income, maternal education, maternal smoking, maternal alcohol use, marital status, infant’s sex, race/ethnicity, gestational age at delivery, mode of delivery, birth weight, batch effects, and estimated nucleated cell types  in all analyses. Statistical procedures for quality control and epigenome-wide analysis of DNA methylation are included as eMethods in the P value distributions are shown in eFigure 1 in the All hypothesis tests were 2-sided. We specified an a priori significance threshold of P = .03) and mean (SD) birth weight–for–gestational age z score (0.38 [0.91] vs 0.11 [0.96] P = .004) relative to infants of fathers with BMI less than 25. We noted a significant positive correlation (r = 0.32) between paternal and maternal BMI, and maternal BMI differed significantly between the group with paternal BMI greater than or equal to 25 and the group with BMI less than 25 . Positive correlations between BMI of both parents were also observed after dichotomizing the study population by maternal BMI less than 25 vs greater than or equal to 25 and, similarly, in the subsets with paternal BMI less than 25 vs greater than or equal to 25. Sample sizes according to maternal and paternal BMI less than 25 and 25 or greater are presented in eTable 2 in the A total of 429 father-mother-child triads had complete data, including paternal and maternal BMI and cord blood DNA methylation; 107 and 400 participants had DNA methylation data from blood samples available for analysis from early childhood (2.9-4.9 years) and midchildhood (6.7-10.5 years), respectively. Demographic information is summarized in q less than .05 (P = 3.13 × 10−8). This CpG is located in an uncharacterized complementary DNA (cDNA) generated from human testis (AK097528); the nearest known gene is transcription factor AP-2 gamma . Moreover, methylation at cg17206978 was also associated with paternal BMI ; this CpG is located in a CpG island within the 200-bp upstream promoter region of centromere protein A (CENPA) on chromosome 2. Gene annotations and effect sizes for the 9 top-ranking CpG sites are shown in We performed a CpG-by-CpG analysis to examine the association of paternal BMI (exposure) and DNA methylation in cord blood (outcome). Because of the correlation between paternal BMI and maternal BMI, we adjusted for maternal BMI as a continuous variable in our primary analyses (model 1). In adjusted linear regression models, DNA methylation at 2 sites (cg04763273 and cg17206978) was significantly associated with paternal BMI after Bonferroni adjustment A. DNA methan .05 . DNA metMMP25 gene was significantly associated with paternal BMI at a Bonferroni-adjusted genome-wide level of significance   to assess contributions of paternal and maternal obesity on the offspring epigenome. Strikingly, no CpG loci were differentially methylated in association with paternal BMI in the subset of mothers with BMI less than 25 . By contrast, among mothers with BMI 25 or greater , methylation at 18 CpG sites showed a significant association between paternal BMI and locus-specific methylation in covariate-adjusted linear models. This suggests that maternal and paternal obesity may act synergistically to influence DNA methylation in the offspring. Among infants born to mothers with BMI 25 or greater, 1 CpG (cg07451886) annotated to the than .05 . Of noteWe next examined reported functional annotations for genes closest to the CpG associated with paternal BMI. Patterns of tissue expression for the genes nearest the differentially methylated CpG are presented in eTable 3 in the q < .05; eTable 4 in the q < .05). These include SP1, SP2, KLF5, and KLF16. Similarly, we performed motif discovery, searching for sequence motifs that are enriched or depleted in the sequences surrounding CpG associated with paternal BMI (eTable 4 in the q < .05), including multiple GATA, TFAP2A, ZNF354C, and others. For CpG identified in the maternal BMI 25 or greater model, 10 motifs were enriched at false discovery rate q less than .001, including MZF1, MEIS1, ETS1, TFAP2A, and KLF5.We next used motifDB to identify transcription factor binding sites in the 2000-bp sequences surrounding each CpG site identified in the primary analyses with matching score of 95% or higher. By comparison with binding sites identified in a background set of 10 000 randomly selected CpG sites from the DNA methylation array, we identified 133 transcription factor binding sites as enriched , cg26544752 , and cg07908498 . Association with paternal BMI for cg04763273 persisted in both the early  and midchildhood  methylation analyses and survived adjustment for multiple comparisons. In the analysis stratified according to maternal BMI 25 or greater, paternal BMI remained associated with early childhood methylation levels at 3 of 18 CpG sites, cg04763273 , cg13872065 , and cg08862162 , and cg04763273 remained differentially methylated at midchildhood. However, none of the early childhood and midchildhood associations in the stratified analysis survived adjustment for multiple comparisons.To determine whether site-specific offspring DNA methylation differences associated with paternal BMI might persist with time, we examined DNA methylation in whole blood during early childhood (age 3 years) and midchildhood (age 7 years) for those CpG identified in the cord blood analysis . Three oARFGAP3 gene was inversely associated with infant birth weight . Methylation at this CpG was also associated with BMI z score at age 3 years, but the directionality of association had reversed . However, analysis of associations with birth weight did not survive adjustment for multiple comparisons.Given that birth weight was greater in offspring of fathers with BMI 25 or greater, and that infant birth weight is associated with many developmentally programmed offspring phenotypes, we examined associations between infant birth weight and DNA methylation at the 9 individual CpG found to be associated with paternal BMI. Cord blood DNA methylation of cg01029450 in the 2-kilobase (kb) promoter region of the ARFGAP3 was significantly associated with paternal BMI and with lower infant birth weight, a risk factor for future cardiometabolic disease. Locus cg04763273, localized to an intronic region on chromosome 20 between TFAP2C and BMP7, was hypomethylated in the newborn period, and remained significantly hypomethylated in early childhood and midchildhood. Together, our data suggest that paternal BMI can have a lasting association with the offspring’s epigenetic landscape in humans. Our data, from a large cohort study, are notable because prior analyses of paternal effects on epigenetic marks have relied on experimental models or have analyzed either a limited number of patients or a few candidate loci. To what extent our observed associations might reflect underlying shared genetic or environmental risk factors is unclear from this cross-sectional analysis.This study found that paternal BMI was associated with altered DNA methylation in cord blood nucleated cells with genome-wide significance, even after adjustment for maternal BMI and key confounders. We identified a potential additive association of paternal and maternal obesity with the infant epigenome based on analyses stratified by maternal BMI. DNA methylation at 21 which examined associations between parental factors and the cord blood epigenome but found that none of the top-ranking genomic loci associated with maternal BMI were associated with paternal BMI. However, paternal BMI was used as a negative control, with analyses restricted to loci found in the primary maternal BMI analyses, and was not examined as a primary exposure. Our results add to the emerging body of literature showing associations of paternal factors with childhood health outcomes. Associations have been discovered between increasing paternal age at conception and higher risk for neurological disorders such as autism and schizophrenia, birth defects , and stillbirth.22 Moreover, paternal height is associated with birth weight (positive correlation), while paternal smoking is associated with lower birth weight, increased risk of congenital heart defects and orofacial clefts, and increased risk of childhood cancers.22 Associations have been found between both paternal BMI and type 1 diabetes and increased risk of obesity and diabetes in offspring.23 From a mechanistic standpoint, these associations may be mediated by shared genetics, nutritional and/or environmental factors, epigenetic signals transmitted by male germ cells, or nongenetic factors such as sperm quality or function. Indeed, impaired sperm parameters have been found to be associated with lower offspring birth weight.24 Together, these data raise the provocative hypothesis that interventions to optimize fathers’ health prior to conception might influence sperm function, epigenetic marks, and health outcomes in their progeny; this will be an important area for future studies.Our findings of concordance between maternal and paternal BMI contrast with those in the Avon Longitudinal Study of Parents and Children,AK097528, a cDNA clone generated from human testis. A second CpG site (cg11868041) located within the same CpG island was also top ranking in the analysis of paternal BMI within the subset with maternal BMI 25 or greater , sortilin-related VPS10 domain containing receptor 3 (SORC3), coiled-coil serine rich protein 1 (CCSER1 or FAM190A), caspase recruitment domain family member 19 (CARD19 or C9ORF89), and NOP2/Sun RNA methyltransferase family member 7 (NSUN7). Moreover, TFAP2C has been proposed as a marker for undifferentiated male germ cells.32 Of note, NSUN7 has been associated with sperm motility and fertility in mice and humans.34 Many of the differentially methylated loci identified in our analysis were near genes with high levels of expression in brain , which is intriguing given that studies of the sperm epigenome before and after endurance training in men have shown that exercise-induced epigenetic remodeling occurs at loci linked to nervous system development and function,35 while weight loss surgery alters sperm epigenetic marks at loci linked to neural control of appetite.14We found that paternal BMI was associated with increased DNA methylation near the transcription start site for CENPA), which encodes a centromere protein with a histone H3–related domain. This pattern is interesting given that cell cycle pathways appear to be sensitive to prenatal perturbations, as altered expression of cell cycle genes has been reported in multiple models of prenatal perturbation, including intrauterine growth restriction, maternal obesity, and paternal high-fat diet.39Another top-ranking differentially methylated locus was centromere protein A  of histones replaced with protamine residues to allow greater compaction of the nucleus for optimal motility.Some of the strengths of our study include the large sample size, the detailed longitudinal assessments throughout pregnancy and childhood, the use of epigenome-wide DNA methylation as outcome (rather than selected candidate genes), adjustment for numerous biological and socioeconomic confounders and covariates, and the analysis for persistence of methylation patterns from birth to age 7 years.44 but it is unclear whether this would also be the case when an individual is reporting on their partner’s measurements. Many other studies examining effects of paternal BMI have similarly relied on mother’s report of father’s height and weight.46 Another limitation is that we cannot rule out an effect of genetic polymorphisms on DNA methylation patterns. However, we did exclude from analysis all CpG probes located within 1 bp of known single-nucleotide polymorphisms. It is also reassuring that the scatterplots for the associations between paternal BMI and DNA methylation at individual sites were not bimodal, as might be expected if the differences were due to differences in single-nucleotide polymorphism frequencies. A third limitation is that we were unable to determine the mechanisms by which paternal BMI may influence epigenome-wide methylation patterns in progeny. From a mechanistic standpoint, paternal obesity might influence DNA methylation patterns in infant cells via a signal transmitted in sperm, but we did not have male germ cells available for analysis. Fourth, given the observed strong correlation between the BMIs of mothers and fathers, as well as potential for clustering of dietary and lifestyle risk factors for obesity within the family units, we cannot exclude potential effects of shared influences such as genetics, diet, lifestyle, or environmental exposures that might simultaneously affect paternal BMI, child BMI, and DNA methylation. These potential interactions are depicted in eFigure 3 in the We also acknowledge some limitations to our study. First, paternal BMI was ascertained based on mother’s report of the father’s height and weight and was not directly measured. Self-reported height and weight may be biased by social desirability and other factors,This cohort study found that paternal periconception BMI was significantly associated with epigenome-wide DNA methylation patterns in offspring at birth. We further observed that associations between paternal BMI and DNA methylation in a subset of loci remained detectable at age 3 years and age 7 years, suggesting that paternal obesity may impart a lasting epigenetic legacy. Paternal BMI was also associated with infant birth weight, a risk factor for future cardiometabolic disease. These data suggest that paternal obesity should be viewed as a risk factor for childhood health and disease outcomes.


Results also identified significant correlations between FA and urinary MMP9 in white matter adjacent to sensorimotor regions , as well as a correlation in similar sensorimotor regions when examining ADC and urinary MMP2 levels  as well as FA and urinary MMP9/NGAL complex . A large, diffuse cluster of white matter was identified as having a strong correlation between both ADC  and FA  with urinary NGAL levels. In contrast, no significant association between DTI measurements and VEGF was observed. Results suggest that elevated MMP9 or MMP9/NGAL in UCPPS may be related to degenerative neuronal changes in brainstem nuclei through excitotoxicity, while also facilitating synaptic plasticity in sensorimotor regions.The Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network has yielded neuroimaging and urinary biomarker findings that highlight unique alterations in brain structure and in urinary proteins related to tissue remodeling and vascular structure in patients with Urological Chronic Pelvic Pain Syndrome (UCPPS). We hypothesized that localized changes in diffusion tensor imaging (DTI) measurements might be associated with corresponding changes in urinary protein levels in UCPPS. To test this hypothesis, we created statistical parameter maps depicting the linear correlation between DTI measurements  and apparent diffusion coefficient (ADC)) and urinary protein quantification  in 30 UCPPS patients from the MAPP Research Network, after accounting for clinical covariates. Results identified a brainstem region that showed a strong correlation between both ADC (R In 2007, the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) began using the classification of Urological Chronic Pelvic Pain Syndrome (UCPPS) to group a variety of chronic pain syndromes into a single classification . Chronicet al this association was observed for MMP9, MMP9/NGAL and VEGFR1 in males and for all of the proteins in females [The MAPP Research Network has identified several alterations in brain structure and function using functional MRI –8, volum females .Independent investigations have identified an apparent association between inflammatory or vascular biomarkers and DTI changes suggesting a possible biological link between the observed urinary protein levels and microstructural brain changes in syndromes like UCPPS. Studies have shown that MMPs play a role in angiogenesis and inflammation –18, as wIn the current study, we explored possible neuroanatomical associations between urinary biomarker levels and DTI measurements in patients with UCPPS. We hypothesized that the reported protein biomarker levels are positively correlated with ADC, and negatively correlated with fractional anisotropy (FA), suggesting a neurodegenerative process that may be facilitated by elevated inflammation and its related consequences including tissue remodeling and neovascularization. Additionally, we hypothesize that these correlations will be strongest in spatially localized regions of the brain responsible for pain modulation and central control of micturition, including the raphe nuclei and the locus coeruleus complex.et al [et al [All subjects provided informed written consent to participate in the current study. All consenting procedures and protocols were approved by the institutional review board at each of the participating sites, which included the University of California, Los Angeles (UCLA), and Northwestern University (NU). Detailed inclusion and exclusion criteria for UCPPS subjects for the MAPP study are outlined in Landis et al . All uril [et al .Table 1 displays demographic information and average urine biomarker concentrations for the patient cohort.For the current investigation, a subset of UCPPS subjects with both urinary protein biomarker measurements and high-quality DTI data were included. This cohort consisted of 30 subjects, 17 males and 13 females, an average age of 40 years old (± 13 years standard deviation), and average symptom duration of 11 years (± 4 years standard deviation). Symptom severity was adjusted for by incorporating two primary symptom constructs of pain and urinary severity [Briefly, urine samples were collected at study baseline and at the 6-month and 12-month follow-up visits as part of the Trans-MAPP Epidemiology/Phenotyping study  accordinet al [Urine samples were thawed, aliquoted and assayed. Total protein concentration was assessed using the Bradford method, and monospecific enzyme-linked immunosorbent assays  were performed in duplicate in a double blinded manner for MMP2, MMP9, NGAL, MMP9/NGAL complex, and VEGF proteins according to manufacturer’s instructions. The average protein quantitation across duplicates for each protein biomarker was used in subsequent analyses. A more complete description of urine biomarker acquisition and analysis is available in Dagher et al . Urine pb = 0 s/mm2 images, diffusion weighted images acquired in 60 or 61 directions, a b-value of 1000 s/mm2, echo time/repetition time (TE/TR) = 88ms/9500ms, slice thickness = 2mm with no gap, field of view (FOV) = 256mm, and an acquisition matrix = 128x128 (2mm isotropic voxels).  and the University of California Los Angeles (UCLA), as both used the same scanner model (Siemens Trio 3T) and presented high-quality DTI scans with comparable acquisition parameters including 8 r et al.  and prevr et al. –13.MRtrix package , and registered to the ICBM-DTI 1mm FA atlas using linear (12 direction via FSL FLIRT) and then nonlinear (via FSL FNIRT) registration on the FA images, the transforms of which were then applied to the ADC images.Diffusion MRI scans were corrected for eddy currents and motion using the eddy correct functionality of the FSL Diffusion Toolbox (FDT) as part of FSL  . FA and et al [3dttest++ command (https://afni.nimh.nih.gov/pub/dist/doc/program_help/3dttest++.html) from the AFNI software package . 3dttest++ solves for a linear regression at each voxel, which yields slopes and respective T-statistics for the significance of the association between the voxelwise measure (ADC or FA) and the variable of interest when accounting for the other covariates. The resulting SPMs were thresholded at the voxelwise level, with significance set at P < 0.05 for each covariate of interest. Clusters of significant voxels, defined as contiguous voxels with P < 0.05, that exceeded a size threshold greater than 700 μL  where then used for further analysis. This cluster size threshold is highly conservative (approx. 3-4x larger) compared to previous studies [et al. [2 with respective P-value.The voxelwise associations were evaluated within a white matter mask (atlas thresholded at FA > 0.3), including subcortical gray matter structures such as the basal ganglia and the thalamus, as outlined previously , 34. Staet al ; similaret al . The GLM studies , 34, whi [et al. . For visFig 1A). Quantification of the correlation between average ADC within this cluster and urinary MMP2 concentration showed a strong and significant association . No spatially-specific associations were observed between FA and MMP2 concentration on SPMs.A small cluster (1.07mL) within white matter adjacent to the right motor cortex demonstrated a positive, significant correlation between urinary MMP2 concentration and ADC after accounting for age, sex, symptom duration, BPI severity and ICPI total score , namely a region within the brainstem and white matter connecting the sensorimotor regions, bilaterally. A 959uL cluster within the brainstem, encompassing the dorsal Raphe nuclei (DRN) and the locus coeruleus complex , was identified as having a strong association between MMP9 and ADC after accounting for covariates . Similarly, a 1,005uL cluster within the midbrain was found to have a strong association between MMP9 concentration and FA, after accounting for covariates . The second set of clusters were identified as having a strong correlation between MMP9 and FA within generalized sensorimotor regions, bilaterally . No significant difference in the slope between MMP9 concentration and FA between left and right sensorimotor regions were observed (P = 0.4314).Two distinct regions of interest within the brain were identified as having a strong association between DTI measurements and urinary MMP9 concentration . Specifically, a large (46.0mL) cluster distributed throughout cerebral white matter regions showed a strong positive correlation between ADC and NGAL . Additionally, a large (31.0mL) cluster exhibited a strong negative correlation between FA and NGAL  throughout similar regions of the brain.Diffuse regions of white matter illustrated a strong association between DTI measurements and measures of urinary NGAL concentration . An 916uL cluster within a similar brainstem region was identified as having a significant association between FA and MMP9/NGAL complex . In addition to these brainstem regions, a single cluster within the left sensorimotor region (704uL) also demonstrated a significant association between FA and MMP9/NGAL concentration .Comparable to regions associated with urinary MMP9 concentration, a 965uL brainstem cluster, containing the DRN and LCC, was found to have a strong correlation between ADC and urinary concentration of the MMP9/NGAL complex (volume = 981uL), but this trend was driven by a single outlier patient (red circle), as once this outlier was removed there was no apparent relationship . Equally, we observed an initial association between FA and VEGF concentration within the brainstem (volume = 992uL) that was found to be driven by a single outlier patient .Lastly, we examined the association between urinary VEGF concentration and DTI measurements within the brain. Initial results suggested a strong positive correlation between ADC and VEGF concentration localized to the midbrain . In particular, we found changes in diffusion MR measurements most consistent with degenerative processes, namely an increase in ADC and decrease in FA, in the brainstem and bilateral sensorimotor white matter regions with increasing metabolite concentrations. Additionally, widespread alterations in ADC and FA were associated with changes in NGAL, suggesting elevated NGAL may be associated with a high degree of axonal or neural plasticity. While the goal of the present study was not to determine the mechanism(s) by which brain structure and these specific urinary biomarkers might interact, we provide a number of potential possibilities, outlined below, that may help to explain the underlying interaction and may provide promising paths for further research.The current study identified distinct regions of the brain that appear to be microstructurally altered in proportion to the concentration of urinary biomarkers associated with inflammation, tissue remodeling and/or the vasculature -PK11195 radiolabel PET and DTI measures in multiple sclerosis [Another potential mechanism to explain the observations in the current study may be degenerative changes in the brain resulting from neuroinflammation, particularly due to glial activation, which was recently targeted for imaging in chronic lower back pain patients using positron emission tomography (PET) . Severalclerosis  and in pclerosis , and assclerosis  and stroclerosis . While tThere are several limitations to this study, which limit the conclusions including the small sample size. Out of 259 urinary samples from UCPPS patients used in our previous study  and 45 pIn this study, we observed that specific urinary protein levels were highly correlated with brain diffusion MRI measurements of microstructural integrity. Results suggest elevated MMP9 or MMP9/NGAL in UCPPS may correlate with degeneration of neurons in brainstem nuclei, possibly through glutamatergic excitotoxicity, while also facilitating synaptic plasticity in the cerebral cortex. Both NGAL and MMP9 are known to modulate plasticity in the brain and damage to monoaminergic neurons may explain comorbidities including depression that are often associated with UCPPS. Future investigations exploring the potential association between urine protein biomarkers and brain changes as measured by other neuroimaging techniques are further warranted to validate and expand our findings, including structural and functional MRI analyses focusing on measurement of atrophic changes in the brainstem or motor cortex and their association to MMP2, MMP9 and MMP9/NGAL protein levels.


In the last decade, ancient DNA research has grown rapidly and started to overcome several of its earlier limitations through Next-Generation-Sequencing (NGS). Among other advances, NGS allows direct estimation of sample contamination from modern DNA sources. First NGS-based approaches of estimating contamination measured heterozygosity. These measurements, however, could only be performed on haploid genomic regions, i.e. the mitochondrial genome or male X chromosomes, but provided no measures of contamination in the nuclear genome of females with their two X chromosomes. Instead, female nuclear contamination is routinely extrapolated from mitochondrial contamination estimates, but it remains unclear if this extrapolation is reliable and to what degree variation in mitochondrial to nuclear DNA ratios affects this extrapolation. We therefore analyzed ancient DNA from 317 samples of different skeletal elements from multiple sites, spanning a temporal range from 7,000 BP to 386 AD. We found that the mitochondrial to nuclear DNA (mt/nc) ratio negatively correlates with an increase in endogenous DNA content and strongly influenced mitochondrial and nuclear contamination estimates in males. The ratio of mt to nc contamination estimates remained stable for overall mt/nc ratios below 200, as found particularly often in petrous bones but less in other skeletal elements and became more variable above that ratio. Besides its high throughput, NGS gave rise to analyses of ancient DNA-specific DNA damage to reveal patterns that authenticate ancient DNA. Post-mortem DNA continuously accumulates characteristic modifications, in particular deamination at the DNA fragment ends4. Deamination frequency thus increases over time and can therefore reveal the ancient origin of an aDNA sample3. As a second step required for authenticating ancient human DNA, NGS data allow estimating contamination levels of human DNA directly1. Earlier estimates of contamination levels in NGS data from early modern humans were based on so-called diagnostic positions on the mitochondrial DNA (mtDNA), i.e. nucleotide positions that differ between the sample and a comparative dataset of complete modern human mtDNA sequences from world-wide populations5. Reads exhibiting a different allele at these positions than the majority of reads likely constitute contamination. Today, Bayesian approaches allow even more precise estimates by identifying the proportion of sequencing reads that were considered to be authentic aDNA from the studied individual6.The emergence of Next-Generation-Sequencing (NGS) technologies has substantially advanced the field of ancient DNA (aDNA) research1, contamination estimates for the diploid nuclear genome can only be obtained for the haploid sex chromosomes in male individuals, especially for the larger X chromosome7. For female individuals, most current studies therefore restrict analyses to nuclear DNA sequences with typical aDNA damage patterns and thus sequences of trusted ancient origin, often reducing datasets by an order of magnitude8. Alternatively, female mtDNA contamination estimates are extrapolated to the nuclear level and only female samples with low mtDNA contamination estimates are used for population genetic analysis on the nuclear genome. Whether this extrapolation is reliable, however, remains untested, in particular given that mitochondrial to nuclear DNA ratios can substantially vary between and even within bone samples9, potentially affecting the extrapolation of mtDNA to nuclear contamination levels.While quantifications of contamination levels in the haploid and maternally inherited mtDNA are achieved by measuring levels of heterozygosity11. Endogenous DNA portions in this skeletal element exceed that in other parts of the skeleton by up to a factor of 40012. This reduces the cost of shotgun sequencing for ancient human genomes to sufficient coverage10 and allows obtaining sequences from geographic regions with typically poor DNA preservation, such as the Near East, Remote Oceania and Africa17. The exceptionally good DNA preservation in petrous bones has been linked to the high bone density of the bony labyrinth10, which reaches adult size during gestation18 and shows reduced bone remodeling compared to the surrounding tissue19. These conditions seem to be ideal for DNA preservation. Initial studies suggest low mt/nc ratios for petrous bones20. We hypothesize that these low ratios allow reliable estimates of contamination with human DNA using mtDNA, especially for female individuals.The reliability of this extrapolation may also depend on the chosen skeletal element given differences in endogenous DNA content and relative amounts of mitochondrial and nuclear DNA. Recent work has identified the petrous part of the temporal bone as a particularly rich source of endogenous DNA and thus an ideal candidate for aDNA studiesThis is the first study to explore systematic differences in mt/nc ratios between skeletal elements and classically used negative controls, and the relationship between mt/nc ratios and contamination estimates. We compare mt/nc ratios and overall DNA preservation in petrous bones, teeth and other skeletal elements in newly produced and publically available aDNA datasets. Our data show that DNA contamination estimates in males strikingly vary with mt/nc ratios, and argue that the typically low mt/nc ratios in petrous bones make them ideal for making reliable contamination estimates using mtDNA.6,50 = 20.15, p < 0.001; Teeth ANOVA F3,181 = 25.25, p < 0.001; Bones ANOVA F3,71 = 9.26, p < 0.001), with at least one sample differing from all remaining ones in each skeletal element , therefore combining them for all further analyses.To assess the mt/nc ratio of potential sources for contamination of archaeological samples, we also measured the mt/nc ratio of human contamination originating from plant extracts3,336 = 15.01, p < 0.001). Variation was again similar between teeth and other bones (Tukey HSD post hoc P = 0.75) but significantly larger in these two than in petrous bones (both P < 0.0001).The mt/nc ratios showed significant variation Fig.  between To assess the degree to which mt/nc ratios vary with sample characteristics within sample origins, we tested relationships with endogenous DNA content, age, and mapping stringency. Despite much between-sample variation, mt/nc ratios almost consistently decreased with increasing endogenous DNA content across skeletal elements and sampling origins . Changes in the mapping stringency in terms of mismatches allowed per read  showed some fluctuation in the mt/nc ratio in T1 and T2 teeth, but no directional effect on mean mt/nc ratios .In contrast, we detected no significant relationship between radio carbon dates of the P2, P3, T3 and B2 samples and their loge[mt/nc ratio] between 0 and loge(200) = 5.3 , but strikingly increased with loge[mt/nc ratio] at values exceeding loge(200) = 5.3 . This stability is reflected in the fact that samples with a ratio below loge(200) = 5.3 are located on the bisecting line when plotting MT contamination against X contamination  may be highly misleading. For example, three “other bone” samples in the current study exhibited less than 3% contamination on the mtDNA, contrasting to nDNA-contamination between 16 and 30% measured on the X-chromosomes. All three samples were characterized by high mt/nc ratios spanning 870 to >56.000. For these samples, it is not possible to determine from the distribution of reads on the X and Y chromosome with certainty if they are males with a contaminated X chromosome or if they are females with male contamination. Either way, the contamination on the nuclear level is too high for further population genetic analysis and even in absence of Y chromosomal reads as an indicator of contamination, the extrapolation from the mtDNA contamination level could still underestimate the actual nuclear contamination level in case of high mt/nc ratios. Therefore, we advise to take the mt/nc ratio into consideration if only the mtDNA contamination estimate is used in order to choose female samples to be included in population genetic analysis. An effect of mt/nc ratio on the contamination estimates of mtDNA and nDNA as described above was already suggested in the context of analysing Neandertal nuclear and mtDNAe[200] = 5.3) seem to provide a rather reliable estimate of contamination on the mitochondrial level  did not reveal a particular trend. Allowing more mismatches  resulted in similar values in the ratio than more stringent mapping (-n 0.2 and -n 0.8). We could therefore not detect any effect of the bacterial background in aDNA extracts on the correlation between high mt/nc ratios and lower percentages of endogenous DNA.From these influences of the mt/nc ratio on the contamination estimates, we conclude that the mt/nc ratio should be considered if mtDNA contamination estimates are used to select female samples for nDNA studies. Reasons for the observed variation in this ratio have already been subject of different studies and no clear reason for the elevation of the mt/nc ratio in some aDNA samples compared to modern DNA has been identified. One possibility could be a bias of the mt/nc ratio originating from bacteria, since mtDNA contains features of a bacterial chromosome and bacterial reads from the environmental background might map better to the mtDNA elevating the mt/nc ratio. However, mapping with different stringencies  and teeth (T3), and only a weak trend in other bones (B2). Therefore, we found no indication that different decay rates cause the observed variation in the mt/nc ratio.It has been suggested that the higher ratios in aDNA might originate from better protection of DNA in the mitochondrion due to its double membrane27 in their study of different parts of teeth. Their observation indicates that higher mt/nc ratios in dentine may originate from its higher metabolic activity as compared to enamel and cementum. This assumption, of different metabolic activity resulting in different mt/nc ratios could also be applied to other skeletal elements, the petrous bone in particular. In the cortical part of the petrous bone, bone remodelling is suppressed compared to surrounding tissue by a specific signalling-pathway, and the number of metabolic active osteoclasts and osteoblasts is reduced28. Furthermore, the more compact bone regions contain a higher number of osteocytes, which have lower numbers of mitochondria29. The number of mitochondria in osteocytes decreases from periosteal and endosteal surfaces towards the inner and denser bone parts30. Consequently, sampling the hardest part of the petrous bone results in low mt/nc ratios due low mt/nc ratio in this area ante-mortem. Within the densest parts of the cortical bone displaying low mt/nc ratios, the endogenous DNA is also better protected against the environmental background of bacteria, fungi and other microbes resulting in high percentages of endogenous DNA.Another reason for differences in mt/nc ratios could be higher metabolic activity in particular skeletal elements, as suggested by Higgins and colleaguesante-mortem, which is caused by a low metabolic activity.In conclusion, we showed that the mt/nc ratio is an important value in aDNA authentication. It strongly influences the accuracy of extrapolating nDNA contamination levels from mtDNA contamination estimates. This approach should be used with particular caution if mt/nc ratios exceed 200. Lower mt/nc ratios are often associated with high percentages of endogenous DNA, as typically found in the densest parts of the petrous bones. In this case, low mt/nc ratios are most likely derived from a low mtDNA concentration in this bone region 31. While age can only be roughly estimated depending on developmental stages32. In addition, the discovery of the higher percentage of endogenous DNA in petrous bone compared to other skeletal elements11 constitutes a substantial improvement for the field of aDNA. Our study confirms those findings and shows that mitochondrial to nuclear ratios provide a further argument to extract ancient DNA from petrous bones providing reliable human contamination assessment from both sexes.Temporal bones, including the petrous bones have already proven to be valuable in physical anthropology. The compact structure of the petrous bone is ideal for preservation in archaeological contexts, therefore, it can be used to estimate the minimal number of individuals34, the late Neolithic multiple burial of Muttenz35, the final Neolithic sites in Switzerland Spreitenbach36, Seengen35, Zurzach35 and cave finds from Wartau37  with published datasets (Table 10 (P2) and four from Broushaki and colleagues13 and Gallego-Llorente and colleagues14 (P3). Teeth samples (n = 185) included 36 from the Swiss burials , 88 from Allentoft and colleagues38 (T3) and 61 from Schuenemann and colleagues39 (T4). P1 and T1 from this study originate from the same multiple burial in Oberbipp, Switzerland but due to the comingled nature of the remains an assignment of the teeth to the petrous bones is not possible. Furthermore, the endogenous DNA in the teeth is too low to allow kinship analysis to identify identical individuals. Therefore, it is possible that teeth and petrous bones are from the same individuals. Finally, diverse bone samples from compact bone parts (n = 75) included 13 from Allentoft and colleagues38 (B1), 52 from Schuenemann and colleagues39 (B2) and eight from Günther and colleagues40 (B3) and two from Broushaki and colleagues13 (B3). Fastq files were downloaded from the European Nucleotide Archive. Radio carbon dates were used from Gamba and colleagues10, Broushaki and colleagues13, Gallego-Llorente and colleagues14 and Schuenemann and colleagues39.We combined shotgun data from 76 new ancient DNA samples from the late Neolithic dolmen burial site of Oberbipp in Switzerland41. If necessary, adapters were removed and paired-end data was merged using Clip&Merge41. Mapping was performed with BWA with the mismatch parameter set to 0.01 and a seed length of 1,000. If necessary, PCR duplicates were removed using DeDup41. Mitochondrial to nuclear ratios were calculated by dividing the mean coverage of the mitochondrial genome by the mean coverage of the nuclear genome to take into account the different length of nuclear and mitochondrial genome. Samples with no reads on the mitochondrial chromosome resulting in a ratio of zero as found in some negative controls, plant extracts, and extremely bad preserved teeth samples were excluded.All data were processed with the EAGER pipelineschmutzi22. Mitochondrial contamination was then estimated with a Bayesian approach as described in Fu and colleagues6. Sex determination was performed after Skoglund and colleagues42. X chromosomal contamination in males with more than 0.5-fold coverage on the X chromosome was estimated using ANGSD23.Reads mapping to the mitochondrial genome were extracted from the BAM files and the mitochondrial genome was reconstructed using the software e[mt/nc ratio]  between the sub-samples within each of the three different skeletal elements and controls, followed by Tukey HSD post hoc tests. Second, to assess overall differences in loge[mt/nc ratio] between skeletal elements and controls, we performed linear mixed models as implemented in the lme4-package for R43. Given substantial variation in loge[mt/nc ratio] between different sample origins and studies detected above, this model contained the source study as a random slope factor, allowing between-study variation in the main effect while maintaining the paired nature of samples from the same source. Pairwise Tukey HSD post hoc tests were calculated from this model using the lsmeans package for R44. Differences in the variance of the loge[mt/nc ratio] between skeletal elements and control were tested using Levene’s test, followed by Tukey HSD post hoc tests.Statistical analysis was performed in R version 3.4.3 (R Core Team 2017). We first used one-way ANOVA to assess differences in loge[mt/nc ratio] varies with DNA-content  we constructed a linear mixed model as above with loge[mt/nc ratio] as the response variable, loge[endogenous DNA] as the predictor, and subsample as random slope factor allowing differential regression slopes between subsamples. In addition, we added skeletal element and its interaction with loge[endogenous DNA] as fixed factor to assess slope consistency between skeletal elements. Given that this interaction was clearly insignificant  we removed it from the final reported model. Information about endogenous DNA content was only available for all Swiss and Egyptian samples. An identical model structure was used to assess the relationship between sample age and loge[mt/nc ratio], now with sample age as the main predictor.To assess how loge[mt/nc ratio] between different mapping stringencies were assessed using linear regression. Information about loge[mt/nc ratio] at different mapping stringencies was obtained for the teeth of the Swiss burials sites Oberbipp and Spreitenbach (T1) since they have low endogenous content and a high portion of bacterial background.Differences in loge[mt/nc ratio] we constructed linear mixed models as above, but separate for samples with low (<5.3) and high (>5.3) loge[mt/nc ratio]. Contamination ratio served as the response variable, loge[mt/nc ratio] as the predictor, and skeletal element as a random slope factor allowing differential responses to loge[mt/nc ratio].Finally, to assess how the ratio of mitochondrial over X-chromosomal contamination varies with logSupplementary InformationSupplementary Dataset


There are no existing treatments for the long-term degenerative effects of traumatic brain injury (TBI). This is due, in part, to our limited understanding of chronic TBI and uncertainty about which proposed mechanisms for long-term neurodegeneration are amenable to treatment with existing or novel drugs. Here, we used microarray and pathway analyses to interrogate TBI-induced gene expression in the rat hippocampus and cortex at several acute, subchronic and chronic intervals  after parasagittal fluid percussion injury. We used Ingenuity pathway analysis (IPA) and Gene Ontology enrichment analysis to identify significantly expressed genes and prominent cell signaling pathways that are dysregulated weeks to months after TBI and potentially amenable to therapeutic modulation. We noted long-term, coordinated changes in expression of genes belonging to canonical pathways associated with the innate immune response . Bioinformatic analysis suggested that dysregulation of these immune mediators—many are key hub genes—would compromise multiple cell signaling pathways essential for homeostatic brain function, particularly those involved in cell survival and neuroplasticity. Importantly, the temporal profile of beneficial and maladaptive immunoregulatory genes in the weeks to months after the initial TBI suggests wider therapeutic windows than previously indicated. There is a singular disquieting fact about traumatic brain injury (TBI); thus far, all successful preclinical studies have failed to translate , 2, indiAnother reason for urgency in identifying long-term pathogenic mechanisms is that TBI increases the risk of dementia later in life . In a naWhile acknowledging the dismal state of TBI therapeutics, how do we address this challenge and provide hope for existing TBI survivors? What are the endogenous deleterious and protective signals in the injured brain that could be therapeutically targeted with currently approved drugs to improve recovery and functional outcome after TBI? Is it too late, or are there wider therapeutic windows that provide hope for current chronic TBI survivors? As part of a larger project documenting proteomic, histological, behavioral  and genoOur previous studies , 18 led ad libitum in a vivarium with these constant conditions: light cycle (6: 00–18: 00) temperature (21°C–23°C), and humidity (40%–50%). All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch, Galveston, Texas and conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals . For each post-TBI interval, we analyzed gene expression in hippocampus and cortex of four naïve, four sham-injured and four TBI rats; thus, 84 rats were used for microarray and PCR array analysis.Adult, male Sprague-Dawley rats (250 g–300 g) from vendor Charles Rivers  were housed two per cage with food and water 2: room air (70:30) using a volume ventilator . Rats were prepared for parasagittal fluid-percussion injury (FPI) as previously described [Rats were anesthetized with isoflurane in an anesthetic chamber, intubated, and mechanically ventilated with 1.5%-2.0% isoflurane in Oescribed , 20. Surlater (ThermoFisher) and stored at 4°C or placed into separate empty sterile Eppendorf tubes and fresh frozen on dry ice for 3–5 minutes. After retrieval of the hippocampi, a small “snip” of cortex near the injury site was excised using the Iris forceps and placed into separate sterile Eppendorf tubes containing RNAlater (ThermoFisher) and stored at 4°C or placed into separate empty sterile Eppendorf tubes and fresh frozen on dry ice for 3–5 minutes.Tissue dissections were performed using sterile, RNase-treated iris tissue forceps. The two cerebral hemispheres of the cortex were opened along the cerebral longitudinal fissure and separated with the forceps to expose the hippocampus. Each hippocampus was individually separated from the cortex and midbrain using the same Iris tissue forceps and each were placed into separate sterile Eppendorf tubes containing RNAlater  at 4°C. Tissue was shipped to GenUs Biosystems  for microarray analysis. We have a long standing collaboration with GenUs scientists, who participated in the first MicroArray Quality Control (MAQC) study that demonstrated the inter- and intraplatform reproducibility of gene expression measurements [Microdissected hippocampal and cortical tissue, collected at 24 hours, 2 weeks, 3, 6 and 12 months after FPI, were placed in a 1 ml tube and stored in RNAurements . Thus, ourements , 22. RNAThe filtered gene list was queried for genes differentially expressed (fold-change ≥1.4) in TBI relative to sham treatment. Statistically significant gene expression data (p < 0.05) from the GeneSpring analysis were uploaded into Ingenuity Pathway Analysis (IPA) software (QIAGEN). Core expression analysis was performed for genes 24 hours, 2 weeks, 3, 6, and 12 months post-injury, to identify critical canonical pathways in which genes were enriched and upstream genes that could potentially be master regulators. P-values were calculated based on a Fisher's Exact Test by considering the number of genes that comprised a given process or pathway within our dataset and the number known to be associated with that process in IPA's reference set. The more genes that overlapped in our dataset with the reference set, the more likely the association was not due to random chance. Thus, significant canonical pathways represent processes with an over representation of focus genes from our dataset greater than were expected to show up by chance. Gene Ontology enrichment analysis was used to identify top biological processes associated with differentially expressed genes at each post-injury interval. Qlucore Omics Explorer , a bioinformatics software platform (based on R) that allows a dynamic and interactive visualization of multivariate data by projecting high dimensional data down to lower dimensions, was used for principal component analysis. Microarray data have been deposited for public access in the National Center for Biotechnology Information Gene Expression Omnibus under accession number GSE111452.For validation of gene expression from 24 h, 2 wk, 3 mo, 6 mo, and 1 year microarray results, qRT-PCR was completed using total RNA isolated at GenUs Biosystems. For each post-injury interval, 10–20 differentially expressed genes from the microarray analysis were selected based on the IPA analysis. Total RNA (500 ng) was reversed-transcribed using the High Capacity Kit (Applied Biosystems) following manufacturer’s protocols. Q-PCR was performed on a Roche Light Cycler 96 using Taqman probes (Applied Biosystems) following manufacturer’s protocols. All data collected was analyzed using the Roche light cycler software, a data analysis tool for sample comparison using the ΔΔCT method for calculating the relative quantification of gene expression.For custom qPCR array analysis of one and two month post-TBI rat brains, total RNA was isolated from microdissected hippocampal and cortex tissue using Trizol (Invitrogen) according to manufacturer’s protocol and DNase treated for 30 min at 37°C using Turbo DNase (Ambion). Total RNA (700 ng) was reverse transcribed using the iScript Reverse Transcription Kit (Bio-Rad) according to manufacturer’s protocol. QPCR was performed on a CFX384 Real-Time System (Bio-Rad) using a Sybr-green 384 well custom PCR array plate containing 4 individual 96-well gene groups . The thermo-profile cycling protocol: 2 min @ 95C for 1 cycle, 5 sec @ 95°C and 30 sec @ 60°C for 45 cycles and a melt curve 5 sec/step @ 65°C to 95°C for 1 cycle was used to run PCR plates. Data was collected and imported into the CFX Manager PrimePCR analysis software. Data was normalized to 5 endogenous control genes  and PrimePCR plate controls were analyzed to verify RNA quality and to test performance of reactions. The ΔΔCT method for calculating the relative quantification of gene expression was used to calculate fold changes comparing sham and TBI groups to naïve groups. We previously employed a similar experimental design using pathway-specific PCR arrays to analyze gene expression in laser captured dying and surviving neurons .The microarray analysis was initially performed on microdissected rat hippocampal and frontal cortex tissues from post-TBI intervals 24 hr, 2 weeks, 3, 6 and 12 months . To fillFor statistical analysis of qPCR validation experiments, a Mann-Whitney test was performed to compare differences between Sham and TBI (p<0.05). For one and two months PCR arrays, normalized expression values were log transformed to distribute data normally. A one-way ANOVA was performed on each gene. A Benjamini-Hochberg (BH) correction test was run and a Tukey’s post hoc test was performed on the genes that had a BH q-value of <0.05.The model of parasagittal fluid-percussion injury (FPI) used in our studies recapitulates many important, clinically relevant hallmarks of human TBI  and has Agilent microarray gene expression data for sham and TBI were normalized to the mean expression of the time/age-specific naïve samples. For clarity and to illustrate temporal gene expression (GE) patterns in the TBI groups, hierarchical clustering heatmaps were generated to show post-injury interval-specific GE across all hippocampus  and cortin silico analysis showed that the genes that distinguished sham from TBI rats were associated primarily with immune response, synaptic function and proteostasis and all annotated genes are known to be essential for normal homeostatic brain function [Qlucore Omics Explorer is a data analysis tool that combines powerful statistical and mathematical tools with instant visualization of large datasets with dimension reduction. The instant, interactive principal component analysis (PCA)- a data dimension reduction technique that allows visualization of high-dimensional, multivariate data without the loss of more information than necessary- performed in QOE is an unsupervised  approach that is well suited for this time series gene expression study. Principle component analysis of hippocampal and cortical GE data shows that at each post-injury interval and as long as one year after injury, the TBI rats were clearly distinguishable from sham-injured controls . For thefunction .p-value ≤ 0.05) from across all time points in the ipsilateral hippocampus and cortex in TBI vs sham-injured controls. Biologically relevant changes were assessed in IPA through core expression analysis done for each set of filtered genes, at each discrete time point. The top 25 canonical pathways (CPs) at 24 hr for hippocampus and cortex are shown  that were enriched in canonical pathways relevant to TBI pathophysiology  Signaling”; and, “Complement System”. In the cortex, NF-κB signaling was more prominent than TLR signaling. We used IPA’s Path designer tool to represent these five immune related pathways and their connection to differentially expressed TBI-associated genes in the hippocampus and cortex . AlthougOne great advantage of IPA is the access to the curated literature that underlies the functional annotation of identified genes and pathways. Thus, IPA analysis showed that many genes in these immune-related pathways are hub genes that are essential components of key cell signaling networks. One prominent set of genes that have been shown in preclinical studies to effect acute outcomes after TBI are members of the complement system . The rolBdnf [Bdnf [At 2 weeks post-TBI, in both the hippocampus and cortex, IPA analyses showed a clustering of dysregulated genes in many of the same immune response pathways found altered at 24 hr . NotablyBdnf . The NF-nf [Bdnf .Cd44, Itgb2, Ncf1, and Rap1b 2 weeks post-injury indicates a persistent activation of immune cell populations after injury. One of these genes which codes for the hyaluran receptor protein CD44, a cell-surface glycoprotein involved in immune cell adhesion and migration, has been previously shown to be upregulated up to three months after traumatic injury [In silico analysis indicated that the multiple immune-related genes upregulated in the hippocampus and cortex at 2 weeks are all implicated in critical cell functions. For instance, Rap1b has essential roles in immune function [Among other immune related processes is leukocyte extravasation . It has c injury , 47. In function  and in afunction . This hiCustom PCR array analysis at one and two months post-TBI was helpful in filling in gene expression trends observed at 2 weeks post-injury  such as the dopamine receptors (DR), pathways –S4 FilesAtf3 [Creb5, NgfR and Stat3), which merit closer examination in future studies. Upstream regulator analysis also allows us to identify and explore the interactions of individual hub genes that have biological signicance in TBI. For instance, at both 24 hr and 2 week post-TBI in the hippocampus, we identified TNF as a key regulator of multiple differentially expressed immune-related genes that are implicated in the inflammatory response  evidence of long-lasting dysregulation of homeostatic gene expression, particularly immunoregulatory gene expression; 2) many dysregulated genes are key hub genes involved in essential brain cell functions ; and 3) the chronic activation of both deleterious and beneficial genes up to a year post-injury suggests the existence of a potentially wider therapeutic window than previously indicated.The lack of therapeutic interventions is a longstanding frustration for those who treat TBI. In this regard, a compelling reason for our focus on persistent inflammatory dysfunction is that simply documenting chronic neuropathological changes after TBI is not enough; we need to identify druggable molecular targets in chronic TBI so that we can explore therapeutic options that can be immediately implemented in the existing population of TBI survivors. The persistent, long-term dysregulation of immunoregulatory genes in TBI rats is suggestive of a much wider therapeutic window than previously thought and implies that treatment of chronic TBI survivors with immunomodulatory drugs may be beneficial months to years after their injuries.Neuroinflammation has been extensively documented in animal models of TBI, but with a few minor exceptions, most studies only examined inflammatory effects days to a month only after injury . Our finOne aspect of brain injury that has troublesome implications for the global burden of disease and is insufficiently understood is that TBI itself is a well documented risk factor for age-related neurodegenerative disorders such as AD and PD and psychiatric disorders such as depression , 8. The Ngfr, Stat3, Il-18, HspB1) may provide additional targets for therapeutic modulation. Increased levels of dopamine receptors correlate with brain plasticity [S100b levels have been shown to reduce inflammation in the hippocampus in a rat model of epilepsy, the significant decrease in expression of S100b at one year post-TBI would protect against inflammation [Atf3 also appears to be a protective response Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S1 FileAll time points are shown in separate tabs.(XLSX)Click here for additional data file.S2 FileAll time points are shown in separate tabs.(XLSX)Click here for additional data file.S3 FileGenes for each post-injury interval listed in separate tabs.(XLSX)Click here for additional data file.S4 FileGenes for each post-injury interval listed in separate tabs.(XLSX)Click here for additional data file.S5 FileGO categories for 24hr, 2 week, 3, 6, 12 month post-TBI listed in separate tabs.(XLSX)Click here for additional data file.S6 FileGO categories for 24hr, 2 week, 3, 6, 12 month post-TBI listed in separate tabs.(XLSX)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Lolium multiflorum) and Birdsfoot trefoil (Lotus corniculatus), have been investigated in vegetable oil– and diesel oil–amended soils. A long-term (147-day) greenhouse pot experiment was conducted to differentiate the main focus of the study: physical and chemical effects of oil (vegetable and diesel) in freshly spiked soils via evaluating the plant performance and hydrocarbon degradation. Moreover, plant performance was evaluated in terms of seed germination, plant shoot biomass, physiological parameters, and root biomass. Addition of both diesel oil and vegetable oil in freshly spiked soils showed deleterious effects on seedling emergence, root/shoot biomass, and chlorophyll content of grass and legume plants. Italian ryegrass showed more sensitivity in terms of germination rate to both vegetable and diesel oil as compared to non-contaminated soils while Birdsfoot trefoil reduced the germination rate only in diesel oil–impacted soils. The results of the current study suggest that both physical and chemical effects of oil pose negative effects of plant growth and root development. This observation may explain the phenomenon of reduced plant growth in aged/weathered contaminated soils during rhizoremediation experiments.Petroleum contamination and its remediation via plant-based solutions have got increasing attention by environmental scientists and engineers. In the current study, the physiological and growth responses of two diesel-tolerant plant species (tolerance limit: 1500–2000 mg/kg), Italian ryegrass ( Organic contaminants like aliphatic, aromatic, heterocyclic and asphaltene/tar hydrocarbons are collectively termed as total petroleum hydrocarbons (TPHs) Van Epps . TPHs arSoil used in the greenhouse pot experiment was prepared artificially by mixing sand and loess with a ratio of 9:1. Samples taken from this artificial soil mixture were analysed for moisture content  of both oils. These levels of spiking were selected as this concentration is known to have inhibitory effects on germination and plant growth w/w)T2: artificial soil spiked with edible vegetable oil (2% w/w)T3: artificial soil spiked with commercial diesel fuel  were filled according to the following experimental plan with five replicates for each treatment and arranged in a complete randomized block (CRB) design.Subsequently, each pot was sown with a mixture of grass and legume plants by using 240 seeds/pot of Italian ryegrass (IR) and 60 seeds/pot of Birdsfoot trefoil (BT). In the greenhouse, the following conditions (day/night) were maintained for plant growth: light interval 12 h/12 h, temperature 22° C/15° C and relative humidity 50%/60%. The whole experiment was monitored for 147 days. Pots were watered from the bottom when soils seemed to be drying allowing capillary rise for plants. Pots from all the treatments were provided almost the same amount of water keeping in mind the visual observation of pot drying conditions. The levels of manual watering depend on greenhouse temperature and plan biomass, and watering was done in a completely manual way when needed.Seed germination for both IR and BT was observed until 3 weeks after sowing and presented only at the time of the last evaluation. Seed germination (SG) percentage for both plants was calculated separately for each pot. SG (%) was calculated by using formula SG (%) =  as explained by Luo et al. . ParametTPH (C10–C40) concentrations in soil samples collected at the start of the experiment and at the time of final harvesting were determined employing the ISO method 16703:2004 E  was used as post hoc test.Data analyses for all the plant and soil parameters were done using Microsoft Excel and SPSS software packages. The results were calculated and interpreted by analysis of variance (ANOVA), and mean values ± standard deviations were compared between treatments by one-way ANOVA. After testing homogeneity of variance (Levene’s test), Duncan’s test . A significantly higher shoot biomass for BT was recorded in control soils as compared to oil-amended soils. Moreover, BT grown on diesel fuel–amended soil showed a significantly reduced shoot biomass compared to BT grown on soil spiked with vegetable oil Fig. a. At theDuring the whole course of the experiment, a distinct species shift was recorded , while the control showed a uniform mixture of grass and legume plants. Plants grown in oil-amended soil produced significantly less shoot biomass compared to plants grown in non-contaminated control soil Fig. . In termThe highest total root biomass was recorded for plants grown in the non-amendment control soil followed by a significantly lower value in diesel oil–spiked soil and again a significantly lower value in soil amended with vegetable oil Table .Table 4SThe SPAD values of Italian ryegrass generally showed the highest values in the control compared to plants grown in oil-amended soil Table . At the Ci − Cf)/Ci], where Ci stands for the TPH concentration at the initial stage (at the time of seed planting and after soil stabilization) and Cf shows the TPH concentration at the harvesting stage (after 147 days) of experiment. While comparing the TPH concentrations at two mentioned time slots, a substantial decrease (more than 40%) was recorded at the harvesting time.Analysis of hydrocarbons was performed in all the variants, but the removal percentage was only calculated for diesel-spiked soil due to negligible concentrations of hydrocarbons in non-contaminated and vegetable oil–spiked soil. The removal percentage was calculated by a formula as explained in Hussain et al. : 100 × [Within the scope of phytoremediation, rhizoremediation is gaining enormous attention  resultant by the addition of vegetable oil may also be responsible for reduced root development. High water repellence creates compacted soils (high soil strength), a physical stress, which may also influence root elongation Gregory . These cOur results show that there are differences among species in sensitivity to physical effects caused by oil contamination of soil. Germination and growth of Italian ryegrass were strongly affected already by physical effects caused by vegetable oil, while the legume Birdsfoot trefoil showed much less effects that could be attributed to physical effects caused by oil in the soil.2O2), monodehydroascorbate (MDA), superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), phenols, proline and several other antioxidants.Planting IR and BT in mixture showed a 40% reduction in TPH contents of diesel-impacted soils. The favourable remediation abilities of these two plants have been studied extensively for diesel-impacted soils. The mechanism responsible for TPH dissipation is known as rhizoremediation—where joint action of plants and their associated microorganism can degrade petroleum hydrocarbons in the active zone of the rhizosphere (Hussain et al.


Immobilization of proteins has been examined to improve implant surfaces. In this study, titanium surfaces were modified with nanofunctionalized denosumab (cDMAB), a human monoclonal anti-RANKL IgG. Noncoding DNA oligonucleotides (ODN) served as linker molecules between titanium and DMAB. Binding and release experiments demonstrated a high binding capacity of cDMAB and continuous release. Human peripheral mononuclear blood cells (PBMCs) were cultured in the presence of RANKL/MCSF for 28 days and differentiated into osteoclasts. Adding soluble DMAB to the medium inhibited osteoclast differentiation. On nanofunctionalized titanium specimens, the osteoclast-specific TRAP5b protein was monitored and showed a significantly decreased amount on cDMAB-titanium in PBMCs + RANKL/MCSF. PBMCs on cDMAB-titanium also changed SEM cell morphology. In conclusion, the results indicate that cDMAB reduces osteoclast formation and has the potential to reduce osteoclastogenesis on titanium surfaces. Aseptic loosening of implants is a multifactorial process associated  with perTo mitigate this disproportional peri-implant osteoclast activity, bisphosphonates as well as recombinant OPG have been applied to implants in different studies ,10,11,12In peri-implant tissues, however, there is evidence of a biological environment that does not respond to the administration of bisphosphonates with the suppression of osteoclasts . MoreoveUtilizing the same mechanism as that of OPG, the fully human IgG monoclonal antibody denosumab (DMAB) specifically binds primate RANKL with a high degree of efficacy and has a well-established and acceptable safety profile . The safModification of titanium surfaces heralded a new direction to a more favorable interaction between the implant and the periprosthetic environment. Different methods have been developed to immobilize and release bioactive molecules from titanium specimens ,20,21,22Thus, we hypothesize that DMAB immobilized on an endoprosthetic surface could inhibit RANKL-dependent osteoclast formation and activation in the peri-implant environment at very early stages. As an oligonucleotide-mediated technique, nanofunctionalization could facilitate a targeted application of DMAB to reduce aseptic loosening with substantially lower systemic DMAB exposure, which may minimize the risk of treatment-related adverse events. As such, this study’s objective was to examine whether DMAB can be effectively nanofunctionalized on a titanium specimen in a manner that reduces osteoclast formation from stimulated human osteoclast precursor cells.Prior to testing the effect of immobilized conjugated DMAB (cDMAB), we first corroborated the ability of soluble DMAB (2 nM) to successful inhibit long-term osteoclast differentiation in vitro.Human PBMCs were used as osteoclast precursors. The cells were cultivated in the presence of the differentiation stimulators RANKL and MCSF . The lonHistochemical detection of TRAP activity revealed a strongly positive enzymatic reaction of the stimulated PBMCs B. As a rIn addition to the osteoclast differentiation medium, one group received 2 nM of soluble DMAB resulting in a substantially lower enzymatic reaction compared with that of the positive control C.The depicted groups were further examined for resorptive activity on dentine chips. After 28 days, resorption pits were visualized by staining with toluidine blue. The +RANKL/MCSF-stimulated PBMCs formed Howship′s lacunae on several sites of the chips with degraded dentine appearing darkly stained B. The -Rn = 6 donors) and resorptive activity (n = 3 donors) confirmed the eligibility of the depicted control groups for all donors. Moreover, soluble DMAB substantially reduced the accumulation of TRAP stain  and further applied at a concentration of 550 nM to the titanium with the complementary ODN anchor strands. The release of cDMAB was monitored after a standard curve for IgG/DMAB had been established. After the rinsing steps, 83% of the IgG corresponding to cDMAB remained hybridized to the ODN anchor strands. This was followed by an initially pronounced (70% of bound cDMA within 24 h) and then low continuous release, which was observed within 18 days.Evaluation of the osteoclast-specific TRAP5b activity revealed a decreased protein level (P = 0.0513) in the cDMAB group compared to the positive control +CTRL on titanium, and almost reached the level of the –CTRL group on titanium .An enzyme-linked fluorescence assay of total phosphatase activity showed that PBMCs of the +CTRL group on titanium formed large multinuclear cells B. PBMCs Scanning electron microscopy demonstrated that PBMCs after 28 days of culture on titanium had differentiated into giant well-spread cells B, showinEnhanced bony implant fixation can be achieved by increasing new bone formation onto implant surfaces, and also by inhibiting bone resorption around implant surfaces. One therapeutic approach toward those goals is to ‘functionalize’ implant surfaces via the tethering of various bioactive molecules that can stimulate osteoblasts or inhibit osteoclasts. The present study evaluated oligonucleotide-based immobilization of the anti-RANKL antibody DMAB on a titanium surface and its effect on osteoclastogenesis from PBMCs stimulated by +RANKL/MCSF.Oligonucleotide-based nanofunctionalization of titanium surfaces has been successfully applied to immobilize other bioactive molecules , for exaThe progress of implant osseointegration and loosening is a common subject of investigation. Recent studies in a New Zealand White Rabbit model reported that several weeks are required for initial implant osseointegration . The effThe current study analyzed the effects of 28 days of DMAB exposure on the ability of cultured human osteoclast precursors to differentiate into terminally differentiated bone-resorbing osteoclasts. Soluble DMAB (2 nM) affected osteoclastogenesis and abolished the terminal differentiation of osteoclasts. Although TRAP positive multinucleated cells were found, the addition of DMAB prevented the creation of resorption pits.Systemic DMAB therapy increases bone density and strength throughout the skeleton and also carries some risks for adverse events, which is acceptable for most patients with osteoporosis because denosumab substantially lowers the risk of fragility fractures. However, many patients receiving osseointegrating implants do not have low bone mass or increased fracture risk, and for those patients, systemic DMAB therapy would have a less favorable benefit–risk balance. The aim of this study was to establish proof of concept that DMAB can be nanofunctionally conjugated to titanium surfaces. This work represents a potentially important first step in establishing a therapeutic approach that leverages DMAB’s potent osteoclast-inhibiting effects while minimizing systemic DMAB exposure. DMAB has the potential to promote osseointegration even while it remains firmly tethered to implant surfaces by acting as a local ‘sink’ that sequesters RANKL, thereby depriving local osteoclast-lineage cells of an essential proresorptive factor. In contrast, the tethering of most other bioactive molecules, including BMPs, PTH receptor agonists, growth factors, and bisphosphonates, requires their timely release so they can directly bind to  their target cells. DMAB was conjugated to ODN strands and further applied to the titanium with the complementary ODN anchor strands. It was found that DMAB remained hybridized to the ODN anchor strands and was substantially released within 18 days. To test the osteoclast-specific effect of DMAB on the differentiation of PBMCs, the tartrate-resistant acid phosphatase and its osteoclast-specific isoform 5b were used, which constitutes a hallmark of active osteoclasts . As showOne limitation of the study was the impracticability of dentine assays after titanium cultivation, which does not rule out the possibility that some bone resorption may still occur in the presence of immobilized DMAB. However, the complete abolishment of terminal osteoclast differentiation is not the intended goal of implant functionalization, as bone turnover may contribute to bone regeneration and implant fixation ,32. AnotIn summary, we demonstrated that nanofunctionalized DMAB decreased the protein level of the osteoclast-specific TRAP5b and endogenous phosphatases in PBMC treated with M-CSF and RANKL for 28 days. Morphological changes in DMAB-exposed PBMCs cultured in osteoclast differentiation medium suggested an inhibitory effect of titantium-immobilized DMAB on terminal osteoclastogenesis. Collectively, these results indicate that titanium nanofunctionalized with DMAB reduces osteoclast activity by interfering with the RANKL-dependent terminal osteoclast differentiation. This in vitro study suggests that nanofunctionalization of DMAB prior to implant insertion could potentially alter the local periprosthetic milieu in a manner that favors unopposed osseointegration. Local antiresorptive treatment heralds a new direction of improving the endoprosthetic lifespan. The slow release of bioactive molecules in conjunction with improving surface osteoconductiveness may further the goals of preventing aseptic loosening.n = 8) were isolated from purchased buffy coats  to generate osteoclast-like cells. In brief, the leukocyte-rich blood portions were diluted in PBS (1:1) and layered on top of Biocoll Separating Solution . After centrifugation at 2000 rpm for 30 min, PBMCs were isolated from the gradient and purified in several rinse steps using PBS.Human PBMCs from healthy human male donors , 0.6% Penicillin/Streptomycin , and 0.01% Amphotericin B  (base medium). The PBMCs were cultured at 37 °C and 5% CO2 for 28 days and the medium was replaced three times per week with additional factors. Additional factors were added to the PBMCs in four groups: (1) no RANKL, M-CSF, or DMAB (−RANKL/MCSF −DMAB: −CTRL); (2) RANKL + M-CSF, no DMAB (+RANKL/MCSF −DMAB: +CTRL); (3) RANKL + M-CSF + DMAB (DMAB); and (4) RANKL + M-CSF + conjugated DMAB (+RANKL/MCSF + conjugated DMAB: cDMAB).To establish osteoclastogenesis, isolated PBMCs were resuspended in medium and a cell count was performed. The PBMCs were further seeded into 24-well plates as monolayer, onto dentine chips, or onto titanium specimens placed in 24-well plates at the density 6.0 × 106 cells/well. It had been shown in preliminary experiments counting the number of nonadherent cells in the supernatant after 24 h that a 2 h incubation step in the incubator at 37 °C before adding 500 μL of the medium per well was adequate to achieve sufficient adherence of the cells. All cells were supplied with alpha MEM  supplemented with 10% fetal bovine serum , 1% The +RANKL/MCSF group was treated with an osteoclast differentiation medium, which comprised the base medium described above plus 25 ng/mL of recombinant human M-CSF , on days one and three, along with 20 ng/mL of recombinant human sRANK Ligand  in all subsequent media changes. The RANKL/MCSF control group received the base medium only.To preliminarily test the effect of low-dose DMAB on osteoclast differentiation, one group of PBMCs received osteoclast differentiation medium supplemented with a single dose of 2 nM of soluble denosumab .For the subsequent examination of the effect of nanofunctionalized DMAB on osteoclast formation, 550 nM of conjugated DMAB was immobilized onto titanium discs (described below); the DMAB-conjugated titanium discs of 14.7 mm diameter were cultured in osteoclast differentiation medium.n = 6 donors). PBMCs were fixed and stained after 28 days of culture using a commercial kit , and TRAP-positivity was examined under light microscopy.Osteoclast differentiation was monitored by visual histochemistry-based assessment of TRAP staining (Osteoclast differentiation and activity were assessed by measuring the area of toluidine-blue-stained resorption pits on dentine chips. Ivory (from German Customs investigations) was cut into flat disks (9 × 9 × 1 mm) by a diamond-coated cutting band saw . Slices were placed into 24-well plates  after disinfection (70% ethanol for 48 h).n = 3 donors) in the above-mentioned groups and densities. After lysis in Tween 20 , the cells were wiped off the dentine chips with a paper towel. The chips were subsequently immersed in 1% toluidine blue , which revealed resorption lacunae that were quantified with the BZ9000 fluorescence microscope .PBMC cultures were seeded on dentin  were made of the alloy TiAl6V4 and ground with SiC paper P600 (26 μm) . The immobilization of the oligonucleotide anchor strands on the titanium specimens  was perf2O and 0.462 g potassium dihydrogen phosphate and 200 mL deionized water of total pH 6.85), 1.1 µL conjugate solution, and 43.9 µL sterile ultrapure water. The γ-sterilized titanium specimens with the immobilized oligonucleotide anchor strands were completely wetted with the hybridization solution and incubated in the dark at room temperature for one hour. Subsequently, the samples were washed four times in PBS. The last rinse took place with sterile ultrapure water. Cells were cultured for 28 days on titanium in +RANKL/MCSF and −RANKL/MCSF groups. The conjugated titanium cells were treated with osteoclast differentiation medium (+RANKL/MCSF).The hybridization of denosumab–oligonucleotide conjugates to the immobilized anchor strands was carried out directly before the cell colonization of the titanium specimens. The materials used were provided by the Max Bergmann Center for Biomaterials (TU Dresden). Titanium specimens were treated with 50 µL of a 550 nM denosumab conjugate hybridization solution consisting of 5 µL 10× PBS buffer  for quantitative human IgG2 detection. Thus, the amount of DMAB rinsed from the titanium during the hybridization process was measured using an ELISA kit  according to the manufacturer’s instructions.The staining of tartrate-resistant acid phosphatase  in monolTo depict endogenous phosphatases on the titanium test specimens, the cells were fixed analogously to the TRAP staining using a citrate and acetone solution, and after 20 min of air drying, the ELF® 97 Endogenous Phosphatase Detection Kit  was used according to the manufacturer’s instructions.Results were visualized by confocal microscopy (Zeiss LSM 710).Secretion of the tartrate-resistant acid phosphatase isoform 5b into the cell supernatant was measured using the TRAP5b ELISA kit  after 28 days of culture, following the manufacturer’s instructions.After fixation in a 4% formalin solution for 24 h, the cells cultured on the titanium were dehydrated through a graded series of ethyl alcohols (50–96%) and further processed in a Critical Point Dryer. The titanium specimens were subsequently sputter-coated with gold and observed under a scanning electron microscope (Zeiss Typ LEO 1455VP).Inferential statistical comparisons of the three groups were made using one-factorial variance analysis using the GLM procedure of the SAS 9.4 Statistical Analysis System for Windows . All values included in these analyses were averages of at least three replicate measurements. The first global test was an F-test at the alpha level of 5%. Significant failure resulted in post hoc pairwise, two-sided group comparisons with Sidak correction. Corrected p-values below 5% were considered significant.The results indicate that DMAB can be electrochemically coated onto titanium surfaces without losing its functionality. Immobilization of DMAB resulted in a reduction of terminal osteoclast differentiation from M-CSF- and RANKL-stimulated PBMCs. The inhibitory effects of DMAB appear to persist for a sufficient duration to potentially improve early implant osseointegration. The technique allows a one-time, low-dose application of DMAB, thereby minimizing systemic effects of the drug. Collectively, endoprosthetic nanofunctionalization of DMAB capitalizes on the benefits of a local therapy without compromising the efficacy of the treatment and thus has potential as a novel and clinically relevant approach to reduce the risk of aseptic loosening.