Detailing the connection between homeostatic functions of enzymatic families and eventual progression into tumorigenesis is crucial to our understanding of anti-cancer therapies. One key enzyme group involved in this process is the Poly (ADP-ribose) polymerase (PARP) family, responsible for an expansive number of cellular functions, featuring members well established as regulators of DNA repair, genomic stability and beyond. Several PARP inhibitors (PARPi) have been approved for clinical use in a range of cancers, with many more still in trials. Unfortunately, the occurrence of resistance to PARPi therapy is growing in prevalence and requires the introduction of novel counter-resistance mechanisms to maintain efficacy. In this review, we summarize the updated understanding of the vast homeostatic functions the PARP family mediates and pin the importance of PARPi therapies as anti-cancer agents while discussing resistance mechanisms and current up-and-coming counter-strategies for countering such resistance. The human genome strives to maintain integrity in the face of perpetual genotoxic stress enduring both exogenous agents and endogenous factors which threaten the survival of the cell . To defeThe current PARP family consists of 17 enzymes, PARP1 through PARP16  1,2,3,44,5,6,7,8While PARP1, 2, 5a and 5b transfer PAR to their target molecules, PARPs 3-16 transfer MAR, thus distinguishing the proteins as PAR or MAR transferases , with the exception of PARP 9 and 13, which appear to be catalytically inactive, as seen in Although most PARP enzymes are MARTs, the characterization of their functions was elusive for years, largely due to the difficulty of selectively inhibiting them with small molecules ,18. FortAs previously mentioned, the homeostasis of the cell is largely dependent on the activities of the PARP family . PARP 5aIt has been well established that PARP1, 2 and 3 are key players in the cellular response to DNA damage, sharing a WGR domain that regulates the protein’s response through a direct interaction with the damaged DNA ,24. In aSSB can occur due to a number of exogenous and endogenous factors resulting in PARP1 recognizing the SSB and binding to the damaged DNA via its Zn1, Zn3 and WGR domains  [1]. As BER is required to repair DNA that has been subject to base removal and UV damage, among other factors, all of which create an apurinic/apyrimidinic (AP) site . Both PAAfter UV-induced DNA damage, detection and repair can also occur via the nucleotide excision repair (NER) pathway, in which PARP1 has been shown to interact with key members . A key sThe DNA damage sensing ability of PARP1 extends past the gaps of SSBs, capable of detecting the overhanging and blunt ends of double-strand breaks (DSBs) . Cells rNHEJ, the quicker, more frequent and error-prone DSB repair mechanism, is promoted by the MARylator, PARP3, which interacts with proteins in the NHEJ pathway and accelerates the repair . PARP3 iMre11/Rad50/Nbs1) complex to process the ends of the DNA, BRCA1 to control DNA resection and RAD51 loading to mediate strand exchange . Af. Af33]. exchange ,38. Specexchange . The recexchange . MDC1 meexchange . The cruexchange . While tIn response to DNA lesions, the activation of PARP1 leads to the PARylation of serine residues on core histones and facilitates early-phase histone removal at the site of DNA damage ,43. PARyAlthough DNA repair has been in focus, new insights into the role of PARP in replication fork stability and protection are becoming increasingly important in understanding the cellular mechanisms that maintain genomic integrity . ReplicaRecent publications have shown that PARP1 and PARP2 are required to repair DNA DSBs arising from the collision of RFs with unrepaired SSBs . Upon reAnother target of PARP1 recruitment is the RECQ1 helicase, which is critical for the protection of stalled RFs . RECQ1 aPARP10 binding to ubiquitinated PCNA has been shown to be a requirement for restarting stalled forks via translesion DNA synthesis (TLS), which allows synthesis machinery to bypass the fork structures by providing low-fidelity polymerases carrying modified DNA bases . AlthougSimilar to PARP10, PARP14 has been shown to reduce replication stress through the promotion of HR-mediated repair via PCNA association during the S phase of the cell cycle . In the The determination of PARP’s role in the absence of exogenous stress has challenged researchers for years. Recently, an increase in PARP-1 mediated- PARylation events has been detected during the S phase of the cell cycle due to the presence of unligated Okazaki fragments . In eachThe prevention of genomic instability is necessary to restrain oncogenic transformation, cancer development and tumor progression . The impPARP inhibitors (PARPi) are a class of orally administered anticancer drugs that compete against NAD+ for PARP’s catalytically active site . PARP1 iCurrently, there are over 250 clinical trials utilizing PARP inhibition, while PARP inhibitors—Niraparib, Rucaparib, Talazoparib and Olaparib—have been FDA-approved . These iPARPi mechanisms of action have not yet been fully elucidated. However, the loss of PARP’s enzymatic activity has frequently been associated with an inability to modify chromatin and self-dissociate from DNA through auto-PARylation . This mePARP1’s role in the BER pathway through the identification of AP sites and the recruitment of essential proteins is well established as its main role in the resolution of SSBs . In the Although BRCA mutations and HR deficiency leading to irreparable DSBs have been the primary focus of PARP inhibition, the recent uncovering of its mechanistic involvement in the ligation of Okazaki fragments and replication fork progression has led to hypotheses surrounding additional avenues of lethality.PARP1 has recently been suggested to sense Okazaki fragments ,55. In HSynthetic lethality occurs when two interacting genes, which allow for cell viability when individually suppressed, cause cell death when simultaneously perturbed . The abiBRCA1/2 play a critical role in the HR pathway and are highly mutated genes in familial breast and ovarian cancers . In 2005Although BRCA1/2 has been the focus for synthetic lethality, the loss of other vital proteins in the DNA repair and replication pathways has been shown to sensitize cancer cells to PARP inhibitors . SpecifiRPA is an ssDNA-binding heterotrimeric protein that is required in DNA replication and DNA damage repair pathways and is responsible for protecting ssDNA overhangs, recruiting repair factors and activating cell cycle checkpoints . Once inFEN1 has a large role in DNA replication and repair; it is responsible for the cleavage of the 5’ flaps left during Okazaki fragment displacement . ImportaIn addition to FEN1, other proteins involved in the Okazaki fragment maturation process such as Ligase 1 (LIG1) and XRCC1 have been confirmed to be synthetically lethal partners of PARP1 ,76,77,78PCNA-PARP has been introduced as another promising area to pursue in synthetically lethal partnerships. PCNA’s main role is the assurance of replication longevity . NormallThe discovery of synthetic lethality and the continual expansion of proteins that can form these relationships alongside PARP, within or outside of BRCA deficiency, prove exciting for future anticancer therapies. The subset listed above is only a small portion of the partnerships emerging. As we begin to further understand the nuances of replication stress responses and DNA damage repair, the combinatorial diversity of these partnerships and their effect on genomic stability within cancer cells will prove significantly important .PARPi therapy has primarily been implicated in breast and ovarian cancers due to a high frequency of defective BRCA1/2 mutations, resulting in an increased likelihood of synthetic lethality through a loss of efficient HR . DefectiMonotherapy involving Olaparib and Talazoparib has been approved for the treatment of mutated germline BRCA (gBRCA) and HER2-negative locally advanced and metastatic breast cancer (BC) . In a reLike breast cancer, ovarian cancer frequently involves BRCA genes and DDR deficiencies, characterizing up to 50% of high-grade epithelial ovarian cancer patients . Platinu+-like PARP inhibitors in high concentrations have demonstrated decreased survival in normal kidney epithelial cells, while histone-dependent PARPi therapy was active only against the cancer cells, showcasing the clinical possibility of this therapeutic following further testing and development [PARP1 activity and expression are also elevated in clear cell renal cell carcinoma in comparison to normal kidney epithelial cells . Some reelopment ,87,88.Therapies that promote progression-free survival in metastatic castration-resistant prostate cancer (mCRPC) have continued to challenge researchers . In patiLike most cancers targeted by PARP inhibition, people with BRCA1 or BRCA2 mutations are susceptible to pancreatic cancer (PC) . SeveralThe gastric cancer benefit from PARPi has recently been brought to light in both mono and combinatorial therapies, partly due to the extensive amount of DDR and HR deficiencies seen across the cancer . PARPis Non-small cell lung cancer (NSCLC) and PARP inhibition are also being explored clinically . VeliparIt is important to note that varying cancers display and respond to the expression levels of PARP1 in the cell differently. Therefore, biomarkers and pre-screening examinations of the patient are important to understanding the benefit or consequences of PARPi therapy .The most common biomarker used to predict PARP inhibition sensitivity is mutated BRCA1/BRCA2, resulting in non-functional proteins and deficient HR. As previously mentioned, sufficient HR is required for the maintenance of genomic stability when faced with DSBs . Due to Pictured in RAD51, a key member of HR, has a paralog, RAD51C, which can be used as a biomarker for PARPi sensitivity . RAD51C MRE11, an NHEJ and HR protein, plays an important role in the replication stress response . MRE11 iGenerally, HR deficiency (HRD) can be scored and used as a PARP biomarker . The scoIn recent years, replication fork protection and stability defects have come to light as an important factor in PARPi lethality . Many HRThe MYCN oncogene, present in neuroblastoma, induces replication stress by slowing the replication speed and amplifying fork stalling . When MYThe PARP inhibitor Olaparib is FDA-approved to treat gastric cancer; however, phase three trials failed to show a significant improvement in the overall survival of patients . A recenDeadbox helicase 11 (DDX11) is a biomarker of aggressive renal cell carcinoma, with no expression in healthy kidneys and increasing expression as the stage of renal cell carcinoma increases . Once knProstate cancers containing the overexpression of BCL2 have been noted as being a biomarker for PARP sensitivity as well . BCL2 blThe use of biomarkers for PARPi across various cancers and DDR repair insufficiencies is beneficial to determining the best course of treatment, but further elucidation is required to develop a full breadth of biomarkers capable of predicting therapeutic sensitivity. Genomic examinations across patients and cancer types should continue to be investigated.The ability to determine the role and expression levels of various PARP members in different cancer types allows for more direct exploitation when utilizing combination therapeutics and determining which inhibitor may prove most beneficial. Although less understood than other members of the PARP family, PARP4 is thought to be involved in the DDR due to its BRCT domain and can be promiscuously targeted by PARPi therapy due to the shared homology of its regulatory subdomains to those of PARP1 ,109. RecThe tankyrases, distinct from the other PARP family members due to their ankyrin domains, play an impressive role in the Wnt/beta-catenin pathway, which has been proven to be a promising anticancer target . TankyraPARP6 has also recently been characterized by varying expression levels in different cancers, which is hypothesized to be due to a difference in role depending on the tissue type . PARP6 hAlthough PARP7 may be poorly understood, its role in ovarian cancer as a MARylator of alpha-tubulin, leading to microtubule instability and cancer cell motility, indicates the possibility of its inhibition as a new avenue of PARP therapeutics . PARP7 hDespite PARP10’s role in overcoming replication stress, it has also been shown to quell tumor metastasis through the regulation of cell migration . It doesPARP11’s role in stabilizing the nuclear envelope during localization is well known, but it has also recently been linked to the positive regulation of immunosuppressive tumor microenvironments ,120. In PARP14’s role in suppressing replication stress via HR-mediated repair makes it an intriguing topic of discussion for inhibition . RBN0127Interestingly, PARP16 has been shown to be a target of the PARP1 inhibitor Talazoparib when used as a therapeutic for small cell lung cancer, showing the range of Talazoparib and depicting a larger view of its mechanism of action . In ovarAlthough the role of the PARP family and the therapeutic value of its inhibition in cancer are continuously being uncovered, the clinical promise has been dampened by the occurrence of drug resistance ,125. TheInterestingly, BRCA-independent restorations of HR including the repression of the NHEJ pathway have also been shown to result in PARPi resistance . 53BP1 iThe upregulation of RAD51 and its subsequent foci formation have also been documented in PARPi resistance, indicating its ability to compensate for the loss of BRCA in HR ,130. On The restoration of replication fork protection has also been shown to confer resistance to PARPi therapeutics by improving genomic stability ,134. It Another proposed mechanism of PARPi resistance is the restoration of PARP1 signaling through the loss of PAR glycohydrolase (PARG), which is responsible for the degradation of PAR chains . It has Various other mechanisms of resistance such as point mutations in PARP1 leading to reduced PARP trapping, the upregulation of drug efflux pumps such as Abcb1a/b and the dysregulation of signaling pathways such as PI3K/AKT have been documented as well ,138,139.Resistance to PARPi is a growing challenge across many forms of cancer and highlights the many ways in which we are constantly at tug of war with the disease. Although resistance is a concern and may lead to a loss of therapeutic effectiveness, it may also give insight into the development of novel combinatorial therapeutics as well as aid in understanding the most effective ways to incorporate PARPi as an anticancer agent.To overcome PARPi resistance, the mediation of synthetic lethality through mechanisms such as immune checkpoint inhibitors, cancer-dependent target therapies, cell combination and DDR inhibitors is becoming increasingly important .The role of immunotherapies in cancer treatments is being vastly investigated. Particularly, the advancements in immune checkpoint inhibitors, chimeric antigen receptors and TCR-engineered T cells have been showing clinical promise ,141. IntPARPi has also been implicated in combination with PI3K inhibitors due to the role of PI3K in oncogenic signaling, leading to cancer cell proliferation and survival ,146. PARThe topic of HDAC inhibitors in the fight against PARPi resistance is part of a large area of focus surrounding the combination of PARPis with other DDR proteins with roles in HR . HDAC inThe regulation of the cell cycle by ATR and CHK1 kinases is crucial for managing replication stress, working to arrest the cell cycle and contributing to fork protection and proficient HR repair . The impThus far, the majority of clinical PARP inhibitors have targeted PARP1, which affects PARP1’s role in the detection and resolution of DNA damage ,18. Due Similarly, the available biomarkers for PARPi account for only a fraction of the possible mutations and interactions that gauge for therapeutic sensitivity. Further biomarker testing across cancer types and within specific patients should be researched to better highlight the targets and cellular responses of PARPi. Biomarkers for specific PARP family members should also be addressed to bring the scope of potential PARPis throughout various cancers to light. As research on the development of selective inhibitors continues, it will be crucial to have data pertaining to cancer signatures that may show the best clinical response.As resistance mechanisms opposing PARPis’ therapeutic potential continue to emerge, the optimization of combinatorial PARPi therapy as a counter mechanism is needed now more than ever. Recently, PARPi has been shown to sensitize tumor cells to immune checkpoint inhibitors through the modulation of the tumor microenvironment in ovarian cancers, hinting at the therapeutic value this combination could have . SimilarThrough years of research, the role of the PARP family as a homeostatic regulator and genome protector has been extensively examined. The enzyme family’s diverse range of functions includes regulating replication, detecting and fixing DNA damage and responding to replication stress . Due to


The study of uterine leiomyomata (fibroids) provides a unique opportunity to investigate the physiological and molecular determinants of hormone dependent tumor growth and spontaneous tumor regression. We conducted a longitudinal clinical study of premenopausal women with leiomyoma that showed significantly different growth rates between white and black women depending on their age. Growth rates for leiomyoma were on average much higher from older black women than for older white women, and we now report gene expression pattern differences in tumors from these two groups of study participants. Total RNA from 52 leiomyoma and 8 myometrial samples were analyzed using Affymetrix Gene Chip expression arrays. Gene expression data was first compared between all leiomyoma and normal myometrium and then between leiomyoma from older black women (age 35 or older) and from older white women. Genes that were found significant in pairwise comparisons were further analyzed for canonical pathways, networks and biological functions using the Ingenuity Pathway Analysis (IPA) software. Whereas our comparison of leiomyoma to myometrium produced a very large list of genes highly similar to numerous previous studies, distinct sets of genes and signaling pathways were identified in comparisons of older black and white women whose tumors were likely to be growing and non-growing, respectively. Key among these were genes associated with regulation of apoptosis. To our knowledge, this is the first study to compare two groups of tumors that are likely to have different growth rates in order to reveal molecular signals likely to be influential in tumor growth. MED12) Uterine leiomyoma is a common, benign, monoclonal, diploid smooth muscle tumor with a frequent mutation in mediator complex subunit 12  was the most highly up-regulated gene in leiomyoma compared to myometrium, consistent with studies conducted by Tsibris DPT) and various isoforms of the fibrillar  and nonfibrillar collagen genes (IV and VI) in leiomoyoma compared to myometrium, findings that are also consistent across other microarray studies ATF3), alcohol dehydrogenase 1b, EGF containing fibulin-like extracellular matrix protein 1, FOS, aldehyde dehydrogenase 1a1and prostaglandin endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase 2 (COX-2)). We also saw significantly lower expression of MED12 in tumor compared to myometrium, though the fold change was small. This is consistent with disruption of this gene by a somatic mutation commonly found in leiomyoma tumors A total of 9516 probe sets were found to be differentially expressed, with fold changes ranging from −14.60 to 73.93 with 1044 probe sets having a fold change greater than 2. The PCA plots using these differentially expressed genes see  suggest COX-2. Genes selected correlated with and supported the direction and relative significance of genes increased or decreased in the microarray analysis, indicating our methodology was robust (COL4A3) and estrogen receptor 2 (ESR2), which had two probes on the microarray chip with opposite directions of expression. We also found the down-regulation of genes involved in prostaglandin synthesis, a finding also reported by Arslan, et al., 2005 To verify expression, the same samples were analyzed for gene expression using RT-PCR, including s robust . The onlIGF1 and prolactin and also included previously undisclosed signaling pathways including axonal signaling, stathmin1, glioma signaling and Hepatocyte Growth Factor . The pathway analysis identified SNAIL2, a mediator of epithelial-mesenchymal transition ERBB2 was also linked to up-regulation of CCNB2, the gene encoding cyclin B2, which was also up-regulated in our gene expression analysis. In animal models, knock-out of erbb receptors result in malformed hearts, and inhibition of erbb2 in adult mice results in myocardial dysfunction and inability of the cardiomyocyte to contract ERBB2 expression results in the inability of myocardiocytes to contract or appropriately differentiate. A similar mechanism may be at play in smooth muscle cells of leiomyoma where the decreased or absent gene expression of ERBB2 underlies a defective contractile and differentiation signal. Particularly intriguing is the question as to what controls the expression of this gene. In leiomyoma, IPA shows a network connection between ERBB2 and DPT, a relationship previously undisclosed in studies of uterine leiomyoma or, to our knowledge, in studies of breast cancer.According to the IPA analysis, one of the top 5 significant network signaling pathways in leiomyoma compared to myometrium was centered on down-regulation of ERBB2 . Most ofIn summary, these results of the microarray analysis from leiomyoma compared to myometrial samples collected from the FGS show robust and consistent results with previously published studies and verified by RT-PCR. Given the validity of the sample set, we next sought to expand these analyses by comparing gene expression in tumors classified as growing and non-growing.p = 0.01. Thus, tumors from black women aged 35 and older (“older black women”) were compared to tumors from white women of age 35 and older (“older white women”) to identify molecular factors that might reflect differential growth.For the tissues available for this microarray study, not all age groups were equally represented or reflective of the age groups in the initial analysis done in the FGS DPT, which was significantly down-regulated in both the comparison between the two leiomyoma groups and in the comparison of all tumors to myometrium. Other genes found to be down-regulated in tumors from older black women compared to tumors from older white women included other matrix-related genes such as fibulin 1, calcium related migratory and endocytotic molecules netrin 1 and stoning 1, and genes involved in metabolism such as pyruvate dehyrogenase kinase isoenzyme IV. Genes significantly up-regulated only in the comparison of the two leiomyoma groups included muscle specific carbonic anhydrase III, which is important in buffering acid-base balance The individual genes in this comparison constituted distinct sets of up-regulated or down-regulated genes that generally differed from those that were differentially expressed between leiomyoma to myometrium  and 4. TRIG-1 pathways associated with a variety of early immune-mediated responses as well as stem cell and cancer cell survival responses  compared to leiomyoma from older white women (more likely to be non-growing) were interferon signaling and retinoic acid mediated apoptosis signaling, granzyme B signaling, and esponses . The ideVHL, a master regulator of a cells response to hypoxia, among the top five significant networks in leiomyomata from older black women (“growing” tumors)  . BecausePARP1, the other on the down-regulation of EGFR , beta 1subunits, MED4, MED19 and MEDIATOR complex. Recently, MED12 mutation was reported in a high percentage of leiomyoma MED19EGFR was only identified in the leiomyoma to leiomyoma comparison, while ERBB2 was identified only in leiomyoma compared to myometrium. The differentiation of these genes suggests a refinement of their roles in tumorigenesis.Two other networks identified as significant in our data have been noted to be important for breast cancer. The first is centered on up-regulation of  of EGFR . PARP1 hThe study of uterine leiomyoma presents a unique opportunity to expose possible fundamental characteristics about the molecular and physical heterogeneity of hormone-dependent tumor growth with relevance to hormone-dependent malignant cancers. Previous gene expression studies of uterine leiomyomata have compared tumors to matched myometrium often with sub-classifications of leiomyoma based on size, location, or menstrual cycle phase to combine groups assumed to have similar characteristics The contributions of our analysis were to identify genes and signaling pathways potentially involved in tumor growth based on statistical modeling results of a detailed MRI study of tumor volume changes over time. Indeed, the signaling pathways identified in the comparison between tumors from older black and older white women are well-recognized in tumorigenesis, particularly those involved in transcriptional regulation and hypoxia, but had not been defined in any other leiomyoma gene expression comparisons. The fact that the comparison is between two ethnic groups may imply a genetic contribution to the data sets, yet, does not subtract from the basic premise that specific genes identified in these comparisons likely play a role in tumor growth.MED12 down-regulation was identified in the comparison of tumors and normal tissue, while the Mediator complex and subunits MED4 and MED19 were specifically identified in the IPA analysis of the older black/older white comparisons of leiomyoma. These findings suggest there may be specificity to global transcriptional regulation and epigenetic controls relating to growth. In contrast, IGF1R and related signaling pathways, classically defined as growth promoting, were not differentially expressed between the two leiomyoma groups, but were up-regulated in leiomyoma compared to myometrium as in other studies of leiomyoma The relevance of genes and pathways identified by this approach is both supported by and substantiates experimental data. For example, Canonical pathways identified in the comparisons of the two leiomyoma groups all related to processes of cell survival consistent with the idea that leiomyoma growth is promoted by an overall inhibition of apoptosis pathways. Contribution of cell survival pathways, as opposed to cell proliferation, is consistent with the generally low mitotic index of leiomyoma. While there is no question that much of the mass of a leiomyoma is due to the collagenous extracellular matrix, we propose that the increase in cellular mass of leiomyoma in older black women most likely to be growing is due to an accumulation of cells that are resistant to tissue and cellular based death signals rather than due to an accumulating mass of actively proliferating cells. Alternatively, a fundamental molecular trigger in regressing leiomyoma may be induction of apoptosis and death signals, rather than due to inhibition of cell proliferation.CAIII), an enzyme which serves to buffer the cellular acid-base balance (pH regulation) . Five micrograms of total RNA from each sample were labeled using the bioarray high-yield transcript kit according to manufacturer's conditions . Labeled RNAs were hybridized and washed according to manufacturer's recommendations . Initial gene expression data analysis was done using Microarray Suite 5.0 software (Affymetrix 2001). All arrays were normalized to a trimmed mean transcript signal level of 500 counts .The log base 2 expression data (perfect match only) were normalized using the systematic variation normalization method Ingenuity Systems, Redwood City, CA, USA). The IPA software is based on computational algorithms of the connectivity from information obtained within the IPA database. IPA analysis accounts for the type of chip used with a score assigned to rank networks according to their relevance to the gene list provided. Canonical pathways were determined by analyzing a ratio of the number of genes that map to the pathway, divided by the total number of genes in the pathway that are represented by the chip probes, and the p value calculated by Fisher's exact test to determine the probability that the association was due to chance alone.Genes that were found significantly different in a pairwise comparison were further analyzed for canonical pathways, networks, transcription factors and biological functions using the Ingenuity Pathway Analysis (IPA) software   . SoftwarFigure S1Principal component analysis plot of the gene expression data of leiomyoma samples  and normal myometrium samples . Left panel is based on all probes on the chip and the right panel is based on differentially expressed probes.(TIFF)Click here for additional data file.Figure S2Principal component analysis plot of the gene expression data of leiomyoma samples from aged black women  and aged white women . Left panel is based on all probes on the chip and the right panel is based on differentially expressed probes.(TIFF)Click here for additional data file.Figure S3Heat map based on probes determined to be significantly different between the aged black and aged white. The data (probe-wise) were standardized to a mean of 0, standard deviation of 1 and then then clustered using the cosine correlation as a dissimilarity measure and average linkage for hierarchical grouping.(TIFF)Click here for additional data file.Table S1Genes found significantly up-regulated in uterine leiomyoma compared to myometrium from FGS.(DOCX)Click here for additional data file.Table S2Genes found significantly down-regulated in leiomyoma vs. myometrium in FGS.(DOCX)Click here for additional data file.Table S3List of all differentially expressed genes between leiomyoma and myometrium in FGS.(XLS)Click here for additional data file.Table S4Quantitative gene expression of a priori selected genes in leiomyoma compared to expression from microarray analysis.(DOCX)Click here for additional data file.Table S5Comparison of up-regulated genes in leiomyoma compared to myometrium from various studies.(DOCX)Click here for additional data file.Table S6Comparison of down-regulated genes in leiomyoma compared to myometrium from various studies.(DOCX)Click here for additional data file.Table S7Taqman Primer Probe Sets.(DOCX)Click here for additional data file.Table S8Custom-designed primer probe sets.(DOCX)Click here for additional data file.


According to the American Cancer Society approximately 242,000 new cases of prostate cancer will be reported in 2012, making it one of the most frequently diagnosed cancers in men. Moreover, with 1 in 9 men diagnosed with prostate cancer anticipated to die of the disease, prostate cancer is also the second-leading cause of cancer death in men. Although several new treatment options for men with advanced prostate cancer have recently become available, none are as yet curative.i.e., pharmacological depletion of androgens) has long been a mainstay for treatment of men with advanced prostate cancer. However, while androgen deprivation initially leads to tumor regression, ultimately it leads to a highly aggressive form of the disease, which has been called “castration-resistant prostate cancer” (CRPC) to indicate its continued dependence on androgen receptor signaling despite the absence of androgens [Because of the necessity of androgen receptor signaling for all aspects of normal prostate growth, as well as for prostate tumorigenesis, androgen deprivation therapy  model of CRPC and in vitro studies using human prostate cancer cell lines to investigate the consequences of dual targeting Akt/mTOR signaling, with an inhibitor of Akt, namely, MK-2206, and one that targets mTORC1, namely ridaforolimus (MK-8669) (Figure in vivo in the GEM models and in the human prostate cancer cell lines in vitro.Consequently, in our recent study we asked whether combined inhibition of upstream and downstream nodes in the Akt/mTOR signaling pathway is effective for suppressing CRPC . We usedWhy would dual targeting of two kinases in the same pathway yield improved results compared with targeting the pathways individually? In part, this reflects the fact that inhibition of mTORC1 alone has limited efficacy, in part due to feedback activation of PI3K/Akt signaling (Figure How might these findings be applied in the clinic? Currently, several studies have described a crosstalk between the PI3K/Akt/mTOR and AR signaling pathways. In particular, it has been suggested that castration-resistant tumors compensate for reduced AR signaling by activation of Akt/mTOR signaling, which highlights the therapeutic potential of combining inhibition of Akt/mTOR pathways with androgen receptor pathway inhibition. Indeed, Carver et al have suggested that inhibition of the PI3K pathway restores AR signaling in PTEN-deficient prostate cells, and that feedback regulation between AR and PI3K pathways may be reciprocal .Thus, based on recent findings by us and others, we propose that a therapeutic approach using dual inhibitors of Akt/mTOR signaling in combination with inhibitors of androgen receptor signaling, such as Abiraterone Acetate or Enzalutamide, may effectively block androgen signaling while simultaneously inhibiting a Akt/mTOR signaling and therefore an effective treatment course for CRPC. We await the results of the relevant clinical trials.


Rivaroxaban is an oral, direct Factor Xa inhibitor approved in the European Unionand several other countries for the prevention of venous thromboembolism inadult patients undergoing elective hip or knee replacement surgery and is inadvanced clinical development for the treatment of thromboembolic disorders. Itsmechanism of action is antithrombin independent and differs from that of otheranticoagulants, such as warfarin (a vitamin K antagonist), enoxaparin (anindirect thrombin/Factor Xa inhibitor) and dabigatran (a direct thrombininhibitor). A blood coagulation computer model has been developed, based onseveral published models and preclinical and clinical data. Unlike previousmodels, the current model takes into account both the intrinsic and extrinsicpathways of the coagulation cascade, and possesses some unique features,including a blood flow component and a portfolio of drug action mechanisms. Thisstudy aimed to use the model to compare the mechanism of action of rivaroxabanwith that of warfarin, and to evaluate the efficacy and safety of differentrivaroxaban doses with other anticoagulants included in the model. Rather thanreproducing known standard clinical measurements, such as the prothrombin timeand activated partial thromboplastin time clotting tests, the anticoagulantbenchmarking was based on a simulation of physiologically plausible clottingscenarios. Compared with warfarin, rivaroxaban showed a favourable sensitivityfor tissue factor concentration inducing clotting, and a steepconcentration–effect relationship, rapidly flattening towards higherinhibitor concentrations, both suggesting a broad therapeutic window. Thepredicted dosing window is highly accordant with the final dose recommendationbased upon extensive clinical studies. The blood coagulation cascade is a complex process, involving both an intrinsic andan extrinsic pathway [1]. TheRivaroxaban is an oral, direct Factor Xa inhibitor approved in more than 100countries worldwide, including the European Union and Canada, for the prevention ofvenous thromboembolism after elective hip or knee replacement surgery in adultpatients, and is in advanced clinical development for the treatment ofthromboembolic disorders. Rivaroxaban only targets one factor within the coagulationcascade, Factor Xa, and its mechanism of action is antithrombin (AT) independentA computer model for blood coagulation has been developed, based on several publishedmodels in vivo coagulationis affected by blood flow, which leads to an exchange of proteins between the clotand the fresh blood pool. The action of calcium ions  were indirectly included in the rateconstants and kinetic parameters, and phospholipid membrane-binding sites weredirectly included in the model kinetics. A portfolio of drug action mechanisms andpharmacokinetic profiles was also included in the model: rivaroxaban and DX-9065a(direct Factor Xa inhibitors), warfarin, enoxaparin and ximelagatran. The model wasadjusted to accurately represent both preclinical information, such as directlymeasured dissociation constants, and clinical measurements such as the commonclotting tests, prothrombin time (PT) and activated partial thromboplastin time(aPTT). Some additional aspects of the coagulation cascade will be developed infurther improvements to the model, including: thrombocytes and their action;fibrinolysis and spatial thrombus properties.The model was built to cover several aspects of the coagulation cascade, includingthe extrinsic pathway (initiated with the triggering of tissue factor[TF]), the intrinsic pathway (initiated with activation of Factor XII[Factor XIIa]) and the common pathways leading to fibrin generation viathrombin generation. Additional features were also included: inhibition via the TFpathway inhibitor or via AT, and the fact that in vitro test systems.This computer model was used to assess the efficacy and safety of rivaroxabancompared with that of other anticoagulants included in the model, by usingphysiologically plausible clotting scenarios with presumably high relevance forbleeding as well as thrombosis events. The computer model was subsequently appliedto the modelling of the therapeutic window of rivaroxaban by comparing theinhibitory effect of all anticoagulants in these virtual clotting scenarios, whichwere not feasible with laboratory The program package MoBi® Equation 1 represents the one-to-one (stoichiometric) conversion of Factor II toFactor IIa (prothrombin to thrombin). Equation 2 describes the kinetic lawgoverning the rate of conversion: it is proportional to the concentrations ofFactor Xa, Factor II, with k16 being the kinetic constant. Kinetic parametersand initial conditions (concentrations) represent the quantitative informationabout the coagulation system and take into account the impact of the differentstudy drugs see: . KineticIndividual factors and interactions were classified using the well-characterizedextrinsic or intrinsic pathways, because the model was constructed to representthe typical in vitro tests used for investigating the extrinsic and intrinsicprocesses  in one unified computationalrepresentation. Transport reactions taking into account the blood flow were thenincorporated, so that the model was more closely related to the physiological invivo setting.The extrinsic core of the model was adapted from the model by Hockin et al. The intrinsic pathway was adapted from the model published by Kogan et al. Further model extensions were taken from the model developed by Anand et al.The protein C/S system and the coagulation factor adsorption reactions to lipidswere developed on the basis of the model published by Bungay et al. Additional coagulation factor inhibition reactions were introduced based onpublished rate constants on and koff) were used when available.When no direct experimental kinetic data for individual biochemical reactionswere available, values were determined numerically by fitting the modelbehaviour to indirect measurements using PT and aPTT values versus drugconcentrations. A broad set of regimens (doses and schedules) was simulated,both with constant drug levels and dynamic pharmacokinetic profiles to Factor IIa, meizothrombin (mIIa), and the thrombin–thrombomodulincomplex were added into the model , Factor IIa, mIIa, Factor IXa and Factor XIa were addedinto the model  for vitaminK-dependent Factors II, VII, IX and X, as well as proteins C and S based onpublished data The model had to take into account the fact that in vivo coagulation is not onlytriggered by weak TF and Factor XIIa concentrations and propagated by thecoagulation cascade, but is also affected by blood flow and the resultingexchange of proteins between the clotting region  and the fresh blood pool that generally includesnon-activated coagulation factors . The mod−7 cm2/s, averaged from the morecomplex hydrodynamic approach of Anand et al. (crit]), which suppresses a coagulation event fora given trigger scenario, was calculated, and comparisons of the differentαcrit values for the study drugs were a useful model outputto benchmark compounds and dosing schedules .The only species not transported are those bound to immobile tissue or to thethrombus . Coupling was determined by a general coupling constant(α), which controls the overall strength of the coupling, and aFactor-dependent diffusion–convection constant, termed D , individualfactors not listed in The initial concentrations for all modelled coagulation factors and otherproteins are given in Kinetic parameters, including kinetic and diffusion constants, are given in Dilution of plasma by a coagulation reagent was defined as reduced concentrationsof all plasma proteins and factors, as well as added drugs, or as adjustedeffective concentrations for surface-bound factors (e.g. TF). Initial plasmaconcentrations of inhibitors (study drugs) were calculated based onpharmacokinetic data.in vitro dilution, in order toreproduce the procedures generally used for the PT test. The tests requirethat an aliquot of plasma be mixed with an aliquot of coagulation testreagent containing trigger(s), phospholipids, calcium ions and buffer. Toinitiate a PT test, TF 4 nM concentrations were used, together withpreactivated 1% of Factor V taken from All concentrations given in in vitro dilution and to reproducethe procedures generally used for the aPTT test. To initiate an aPTT test,2.2 nM Factor XIa and 50 nM Factor XIIa concentrations were used, togetherwith preactivated 1% of Factor V taken from All concentrations given in in vivo conditions relevant to bleeding risks underanticoagulant therapy and relevant to thrombosis, four trigger scenarios wereconstructed  representing contact with subendothelialtissue was chosen. Prolongation of clotting times under this scenario wasinterpreted as an increased bleeding risk. To investigate efficacy, threetriggers were separately applied, which represent typical situations where massiveclotting should not occur, simulating plasma not in contact with subendothelialtissue. In such scenarios, a significant clotting would raise the risk ofthrombosis. These three triggers are interpreted as a complement to the bleedingscenario.As a safety-relevant strong trigger, a high TF concentration and 30 mg twicedaily (bid) for prevention, and 1.5 mg/kg od or 1.0 mg/kg bid for acute therapy.We obtained corresponding plasma concentrations from the manufacturer'sdata sheets Warfarin validation results are shown in Rivaroxaban 20 mg od was compared with warfarin (INR 1.5–3.0). Clottingtimes were measured as a function of TF concentrations . At low −12 mol/l TF,  showed a dependency on TF trigger strength favourable to the vitaminK-dependent inhibition of the coagulation system . At low The therapeutic window for rivaroxaban found by simulating and ranking a broadportfolio of anticoagulants and scenarios was a total daily dose between 5 mg and 40mg  and 10. After another phase II study investigating the once-daily dosing of rivaroxaban inpatients undergoing total hip replacement After two dose-ranging phase II studies for the treatment of venous thromboembolismAppendix S1Comprehensive reaction list of the coagulation model.(DOC)Click here for additional data file.Appendix S2Initial conditions for all species not being zero (coagulation factors andintrinsic inhibitors).(DOC)Click here for additional data file.Appendix S3Kinetic parameters for the model (based on mol/l and seconds).(DOC)Click here for additional data file.


Chemokines are small chemoattractant cytokines involved in homeostatic and inflammatory immune cell migration. These small proteins have multiple functional properties that extend beyond their most recognized role in controlling cellular migration. The complex immunobiology of chemokines, coupled with the use of subsets of chemokine receptors as HIV-1 and SIV entry co-receptors, suggests that these immunomodulators could play important roles in the pathogenesis associated with infection by HIV-1 or SIV. This review provides an overview of the effects of pathogenic infection on chemokine expression in the SIV/macaque model system, and outlines potential mechanisms by which changes in these expression profiles could contribute to development of disease. Key challenges faced in studying chemokine function in vivo and new opportunities for further study and development of therapeutic interventions are discussed. Continued growth in our understanding of the effects of pathogenic SIV infection on chemokine expression and function and the continuing development of chemokine receptor targeted therapeutics will provide the tools and the systems necessary for future studies of the roles of chemokines in HIV-1 pathogenesis. Chemokines are chemoattractant cytokines that are generally less than 100 amino acids in length and that play major roles in immune cell trafficking during homeostatic and inflammatory immune processes . In addiin vitro in vitro in vitro . All thrin vitro , 54, . This mn .CD26, also known as dipeptidyl peptidase IV (DPP IV) cleaves amino-terminal X-pro dipeptides from selected proteins . ApproxiThe metalloprotease aminopeptidase N (APN), also known as CD13, was recently reported to proteolytically process CXCL11 . APN/CD1Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that perform multiple roles in normal immune responses to infection. Some MMPs are able to proteolytically processes subsets of chemokines, and as with CD26 processing, these modifications can increase or reduce their bioactivities -163. Exareg and increased levels of cellular activation. In addition to their agonist properties on subsets of chemokine receptors, numerous chemokines also antagonize heterologous chemokine receptors and increased expression of chemokines during SIV infection, such as for CXCR3 ligand CXCL11, could lead to exclusion of cells from local microenvironments. Examples of this interesting cross-regulatory property of chemokines include: CXCL9, CXCL10, and CXCL11 antagonism of CCR3 , 127; CXChemokines and their receptors are clearly at the interface between the host and HIV-1/SIV in multiple regards, including viral transmission, viral propagation and chronicity, innate immunity, adaptive immunity, the interface between innate and adaptive immunity, inflammation, and cytokine milieus in immune inductive and effector sites. Additional investigation is necessary to understand the full extent and repertoire of changes to chemokine expression profiles in different tissue compartments within infected hosts. These findings need to be considered within a broader systems biological view that considers not just one chemokine and its receptor(s) and not just the chemoattractant outcomes of this interaction, but considers multiple chemokines and receptors, cross-agonism and cross-antagonism of receptors by multiple chemokines, post-translational modifications to chemokines, and the growing list of functional activities of chemokines beyond control of cell migration. Future studies will need to consider simultaneously the events occurring in multiple tissues and will need to include more sophisticated cell trafficking and homing studies. Coupling these investigations with the intense efforts being directed at developing therapeutic strategies against chemokine receptors could provide new avenues for treatment of HIV-1 infected individuals, and the SIV/macaque model system will continue to provide a robust preclinical model with which to begin to test such strategies.Targeting chemokine receptors in the context of HIV-1 infection and disease can be viewed from multiple perspectives. The first is viral entry and replication and CCR5 antagonists have been developed and are being tested. Another view would be to target the pro-inflammatory effects of increased expression of inflammatory chemokines. Any strategy to intervene in such a way is complicated by the overlapping specificities in the chemokine system .ccr5 suggests that pharmacologic inhibition of the receptor will not lead to severe complications, which has been supported in human trials )n trials . One appn trials ) or syntn trials  and PSC-n trials ) at the n trials , 178. Thn trials , and marn trials . Small mn trials -181. Then trials .reg levels in tissues of SIV infected macaques will reduce or exacerbate disease progression [Manipulation of the chemokine and chemokine receptor system beyond inhibition of viral replication through CCR5 could provide tremendous insight into the contributions of pro-inflammatory chemokines to HIV-1/SIV pathogenesis. Given the consistent observation that CXCR3 ligands are up-regulated in multiple tissues during SIV infection, it would be interesting to inhibit CXCR3 in an attempt to reduce the inflammatory positive feedback loop of host IFN-γ responses, IFN-γ induction of CXCR3 ligands, and CXCR3-mediated recruitment of IFN-γ producing cells . CXCR3 igression , 134.Progress continues to be made in understanding the multiple roles of chemokines in the pathogenesis of or protection from disease caused by HIV-1/SIV infection. Linking basic studies of the changes caused by infection, with basic studies of chemokine immunobiology and clinicopharmacologic studies of chemokine and chemokine receptors, will provide new opportunities to test hypotheses and develop novel therapeutic interventions. As with any immunologically-targeted intervention, the challenge will be to identify the optimal potency and specificity that suppresses that which is pathologic yet maintains that which is necessary for appropriate immune function.


We assessed the relative importance of environmental, biotic, and spatial factors structuring diatom assemblages, and performed separate analyses on different diatom guilds. Our results showed that the diatom assemblages were mainly structured by environmental factors. However, unique spatial and biological gradients, specific to different guilds and unrelated to each other, were also evident. We conclude that biological predictors, in combination with environmental and spatial variables, can reveal a more complete picture of the local vs. regional control of species assemblages in lotic environments. Biotic factors should therefore not be overlooked in applied research since they can capture additional local control and therefore increase accuracy and performance of predictive models. The inclusion of biotic predictors did, however, not significantly influence the unique fraction explained by spatial factors, which suggests low bias in previous assessments of unique regional control of stream assemblages.Stream assemblages are structured by a combination of local  and regional factors . The relative importance of environmental and spatial  factors structuring stream assemblages has been frequently assessed in previous large-scale studies, but biotic predictors  have rarely been included. Diatoms may be useful for studying the effect of trophic interactions on community structure since: (1) a majority of experimental studies shows significant grazing effects on diatom species composition, and (2) assemblages can be divided into guilds that have different susceptibility to grazing. We used a dataset from boreal headwater streams in south-central Sweden (covering a spatial extent of ∼14000 km One of the most frequently studied topics in ecology is the relationship between species distributions and local environmental factors While the effect of biotic interactions and dispersal on species community structure has been strongly emphasised in metacommunity research, applied research has traditionally focused greatly on environmental control of freshwater assemblages, which is also reflected in current bioassessment and management approaches 2), which included information about diatom taxonomic composition, invertebrate grazer abundances (biotic factor), environmental  and spatial factors . The main aims of this paper were to investigate whether local biotic predictors can explain additional variation of taxonomic composition Here, we assess the relative importance of environmental, biotic and spatial factors using diatoms as our model organisms. Diatom assemblages are not only useful for assessing the relative role of environmental and spatial factors The sampling was done in accordance with Swedish national and regional regulations and no permissions were necessary for the field work and collection. The field studies did not involve endangered or protected species.2) is located in the River Dalälven catchment (29000 km2) in south-central Sweden . Stream width, depth, flow and canopy cover were measured at 10 evenly spaced transects. Depth and flow was measured at three points at each transect, while width and canopy cover was measured once at each transect. Canopy cover was assessed using digital pictures taken from the middle of the stream (at the stream surface) pointing upwards. The pictures were manipulated in computer software Image-Tools  so that black pixels represented the canopy and white pixels represented open areas. The percentage of black pixels was then calculated and used as an estimation of mean canopy cover. The number of items of coarse dead wood (diameter>10 cm) and fine dead wood (diameter<10 cm) along the 50 m stretch was also counted. Water samples were collected in August-September 2009. Samples were analysed at the SWEDAC accredited laboratory at the Department of Aquatic Sciences and Assessment (SLU) for conductivity, pH, alkalinity, total organic carbon (TOC), nutrients  and water colour (absorbance at 420 nm) according to Swedish standards 2) and preserved in 70% ethanol. In the laboratory, the animals were sorted against a white background and individuals identified to the lowest taxonomic unit possible . Invertebrates were divided into functional feeding groups (according to www.freshwaterecology.info) to obtain scores describing their feeding mode preferences. These taxa were classified as grazers if they were scored ≥50% grazing. Taxa within the grazer-collector guild were combined into orders  before constructing the biotic predictor matrix. This grouping was done to reduce the risk of overestimating the effect of rare grazer/collector species, without having to exclude them from the analysis. We did not simplify the biotic predictor matrix further, since invertebrate species may respond differently to environmental and spatial factors Benthic invertebrates were sampled at each 50 m stream reach in October (2009) using a Surber sampler (14×14 cm). At each site, 15 subsamples were collected  according to the European/Swedish standard method (SS-EN 14407). Counting about 400 valves captures approximately 85% of all taxa present in the sample if the sample contains a total of 50 taxa 3, while the large guild consist of species having a length of single cells>15 µm, and a volume of single cells>100 µm3. A general description of diatom abundance and diversity across and within the guilds is given in Diatoms were sampled in October (2009) according to the European/Swedish standard method (SS-EN 13946) by scraping biofilm from the upper surface of five cobbles using a toothbrush and pooling the contents into one composite sample. The composite samples were stored in plastic containers (250 ml) and preserved in ethanol (70%). In the laboratory the samples were then treated with Hpcnm in R package vegan forward.sel in R package packforAll statistical analyses were performed in statistical software R varpart in R package vegan 2 values in the results). The significance of each testable fraction in the variation partitioning analyses was obtained by using function rda in R package vegan To assess the relationship between local  and regional  predictors and the structure of diatom assemblages, we used partial redundancy analysis (pRDA)  by using the function The physicochemical variables pH, altitude, shading, and total phosphorus and spatial variables, reflecting large  to medium (PCNM 9) scale patterns, were significantly related to diatom taxonomic composition in the River Dalälven catchment. Of the biotic variables, the abundance of grazing Ephemeropterans and Trichopterans correlated significantly with diatom taxonomic composition .Local environmental (8%) and spatial factors (5%) explained 13% of the diatom taxonomic composition, while the unique fraction explained by biotic factors (2%) was marginally significant (p<0.10). The variance explained jointly by environmental and spatial variables  was 3%. Local biotic and environmental factors jointly explained 11% of taxonomic composition, with another 11% of the variance explained by the shared fraction between all three explanatory matrices. However, none of the explained variation was shared between spatial and biotic factors .Both pH and altitude were consistently related to the taxonomic composition of the diatom guilds. Other important environmental variables in one or more guilds were shading , depth, total phosphorus, and absorbance (large-sized guild). Spatial variables reflecting large-scale structures  were consistently related to the taxonomic composition of all guilds. Medium scale structures  were related to taxonomic composition of the high-growth, small-, and large-sized guilds, while small-scale structures (PCNM 16) were related only to the low-growth guild. Of the biotic factors, the abundance of grazing Ephemeropterans and Trichopterans was consistently related to the taxonomic composition of guilds, with the exception of Trichopterans and the low-growth guild. The abundance of grazing Coleopterans was related only to taxonomic composition of the low-growth guild .Local environmental factors were consistently and strongly related to the taxonomic composition of each diatom guild. Within each guild, environmental factors significantly explained 3–11% of the taxonomic composition and the shared variance explained jointly by environmental and spatial variables  ranged from 1 to 7%. However, the unique amount of variation explained by spatial and biotic factors varied more between guilds. Spatial factors explained 5–6% of the variation in the high-growth, low-growth, and small-sized guild, but could not explain any unique fractions of taxonomic composition in the motile or large-sized guild. Similarly, biotic factors explained unique fractions of taxonomic composition within the high-growth, low-growth, and small-sized guild (4–6%), but not within the motile and large-sized guild. The shared explained variation between biotic and environmental factors ranged from 3 to 9% but basically no variation was shared between diatom spatial structures and biotic factors  .Although observational studies like ours limit the assessment of mechanisms, the individual and collective patterns emerging from environmental, biological and spatial factors, and their shared variance, provided us with more detailed insight into what factors determine the structure of diatom assemblages in boreal headwater streams. The inclusion of the biological component was particularly relevant since it helped to increase our understanding of the potential role of biological interactions at the landscape scale in headwater streams, for which our current knowledge is still limited 2 = 0.22, p = 0.001) and the amount of shared variance between the environmental and biological fraction in the variation partitioning analyses also suggested that the grazer and diatom communities show a similar strong species sorting along acidity gradients in this catchment.Environmental and spatial factors explained unique and significant fractions of diatom taxonomic composition in the Dalälven catchment. Unique biological relationships between grazer abundances and diatom assemblage composition were only marginally significant when all diatom taxa were analysed together. The variance explained by environmental factors dominated over the spatial and biological fractions. This result supports the notion that stream ecosystems and their communities are under strong abiotic control It was recently suggested that spatial structures in species data, originating from dispersal mediated trophic interactions, can appear as unique spatial structures in variation partitioning analyses, and therefore overestimate the fraction explained by regional factors According to our predictions, biotic predictors explained unique and significant fractions of the variation in diatom assemblage composition, but only within specific guilds . Experimental studies lend some support to our findings. For example, grazers have been shown to frequently ingest diatoms growing in the upper layer of the biofilm, decreasing their biomass 2). The finding of pure spatial structures in diatom assemblage distribution is in line with previous studies covering both small In contrast to our predictions, spatial factors also had a significant effect on the structure of diatom assemblages despite the relatively small study area  Overall, our study shows that diatom assemblages in south-central Swedish headwater streams are mainly the result of local control. However, regional  gradients were also evident over this relatively small landscape scale, which may reflect high dispersal effects (mass effects). Most importantly, our study indicates that biological species sorting, which was seemingly unrelated to spatial gradients and guild specific, can be evident also at this scale of observation. Although biotic factors have largely been ignored in stream studies at similar or larger spatial scales, we show that it may be important to assess their effect on species assemblage structure, in addition to environmental and spatial predictors, or we risk underestimating the total local control of species communities. However, the inclusion of biotic predictors did not significantly alter the unique spatial fraction explained which implies low bias in previous assessment of local vs. regional control of stream assemblages.Table S1List over the diatom taxa found in the 30 headwater sites investigated, indicating to which size  and growth-form guild (high/low/motile) each taxon was classified. If information was not available for a taxa, this is indicated as n.c. = not classified.(DOCX)Click here for additional data file.


Autism spectrum disorders (ASDs) are a complex neurodevelopmental disorder that display a triad of core behavioral deficits including restricted interests, often accompanied by repetitive behavior, deficits in language and communication, and an inability to engage in reciprocal social interactions. ASD is among the most heritable disorders but is not a simple disorder with a singular pathology and has a rather complex etiology. It is interesting to note that perturbations in synaptic growth, development, and stability underlie a variety of neuropsychiatric disorders, including ASD, schizophrenia, epilepsy, and intellectual disability. Biological characterization of an increasing repertoire of synaptic mutants in various model organisms indicates synaptic dysfunction as causal in the pathophysiology of ASD. Our understanding of the genes and genetic pathways that contribute toward the formation, stabilization, and maintenance of functional synapses coupled with an in-depth phenotypic analysis of the cellular and behavioral characteristics is therefore essential to unraveling the pathogenesis of these disorders. In this review, we discuss the genetic aspects of ASD emphasizing on the well conserved set of genes and genetic pathways implicated in this disorder, many of which contribute to synapse assembly and maintenance across species. We also review how fundamental research using animal models is providing key insights into the various facets of human ASD. Autism spectrum disorders (ASDs) are a complex set of heterogeneous neurodevelopmental disorders categorized by a triad of key behavioral anomalies. Characteristic behavioral abnormalities consist of restricted interests accompanied by repetitive behavior, deficits in language and communication, and the inability to engage in reciprocal social interactions . Autism http://www.cdc.gov/Features/CountingAutism/). ASD is among the most heritable disorders evidenced by family and twin studies with a concordance rate of 70–90% for monozygotic twins , point mutations, and identification of rare variants in synaptic cell adhesion proteins and pathways are some of the ways researchers are providing insight into the pathophysiology of ASD . It is wA large portion of this review will emphasize the well-conserved sets of genes and genetic pathways implicated in ASD, many of which contribute to synapse assembly and maintenance across different species. Given the complexity and heterogeneity of this disorder, it has proved challenging to unravel the underlying causes of ASD from human clinical population alone. Nevertheless, numerous animal models have been utilized that have enormously contributed toward understanding specific aspects that constitute the spectrum of these disorders.Leo Kanner, a psychiatrist, initially described autism well over half a century ago , 1971. Sde novo or familial. De novo CNVs are more prevalent in causing sporadic genomic disorders  cause Rett syndrome which shows characteristic autistic-like behavior in addition to seizures, ataxia, and stereotypic hand movements . More reEnvironmental contributions and other modulating factors are emerging as potential risk factors for ASD. Heavy metals, parental age, immunological proteins, environmental pesticides and insecticides, and food contaminants are thought to act as modulators of ASD . These fin utero and is associated with placental transfer of maternal autoantibodies to neuronal proteins potentially leading to neuronal dysfunction.Prenatal exposure to certain pesticides and insecticides are known to inhibit acetylcholine (ACh) and γ-aminobutyric acid GABA; . StudiesTable 3).Autism spectrum disorder is a complex disorder with no singular pathology and because of this a collective and collaborative approach is the key to understanding its etiology and design of rational interventions. Studies in animals are aimed at modeling the core phenotypes associated with ASD, including communication and social impairments, restricted interests, and repetitive behaviors in an attempt to uncover the mechanisms that underscore the entire spectrum of the disorder. In this section, we will uncover the wide range of both invertebrate and vertebrate model systems utilized by researchers that have collectively made significant contributions toward understanding the mechanisms that underlie ASD  model. NHP model is relevant for understanding ASD due to its high degree of correspondence to human behavior and their striking homology in the anatomy of neural circuits that mediate social behavior . Neuroligin 3 knock out mice serve as a model for non-syndromic autism . Drosophila studies have advanced our fundamental understanding of some of these human disease gene functions, which show features of ASD. Recent studies in Drosophila have started to unravel some of the key genes, such as Neurexin 1 . Perturbations in synaptic assembly or function are commonly reported in many neuropsychiatric disorders  and one Y-linked (NlgN4Y). The identification of ASD-linked mutations in NLGN3 and NLGN4X was an important finding that implicated these genes in the etiology of ASD  are post-synaptic CAMs localized at glutamatergic or GABAergic synapses . NLGNs hy of ASD  and linkin NRXN; . Some ofbilities . This arlitation . NLGN3 keference . NlgN4 kpatients . Studiessmission . Studiessmission .Drosophila neuroligin 2 (dnlg2), which is the fly homolog of human NlgN3 showed reduced synaptic bouton numbers and synaptic transmission  gene which like its vertebrate counterparts is pre-synaptic and is required for proper synaptic growth and neurotransmission (Neurexins (NRXNs) are predominantly presynaptic CAMs , althougormation . Pre-synsmission .de novo deletions in Nrxn1 in individuals with ASD. Although rare, missense mutations  are glycosyl phosphatidyl-inositol (GPI) anchored immunoglobulin (Ig) superfamily proteins with diverse functions ranging from myelination  to synapwith ASD . Deletiowith ASD . Since CCNTNAP2 encodes CASPR2, a multidomain transmembrane protein that is best known for clustering potassium channels at the juxtaparanodes in myelinated axons  is a member of Neurexin superfamily and is a locus that is significantly associated with susceptibility for ASD . CNTNAP2ed axons . CNTNAP2ed axons . It alsos of ASD . In addiwith ASD . Recent seizures . These mseizures .NrCAM is a CAM that has gene homology to NgCAM and is capable of homophilic cell adhesion as well as heterophilic interactions with other non-NrCAM molecules such as Contactin-1, Contactin-2/TAG1, and Neurofascin . More rede novo translocation deleting CDH18 in ASD  and protocadherins (PCDH) include a large family of CAMs, a number of which are required for synaptic formation and function . CDHs mo8 in ASD . This stchildren . Other Cchildren .SCN1A (Nav1.1), CACNA1C (Cav1.2), KCNMA1 (BK Ca2+), and KCNJ10 (Kir4.1) channels  which belongs to the voltage-gated sodium channel family necessary for axonal conduction and action potential propagation. These transmembrane proteins possess a large pore-forming alpha subunit and two auxiliary beta subunits. This organization is important for allowing sodium ions to move through the axonal membrane to initiate and propagate action potentials. Recently, SCN1A has emerged as the most important gene in epilepsy  and function in synapse biogenesis by trafficking and anchoring synaptic proteins and clustering of membrane-associated proteins. Most importantly, the scaffolding proteins serve to link post-synaptic receptors with their downstream signaling components and regulate cytoskeletal dynamics .Figure 1). Strong genetic and molecular evidence has linked SHANK2 and SHANK3 to the development of ASD phenotypes.Shank protein family is one such synaptic scaffolding family of proteins that includes Shank1, Shank2, and Shank3. They have multiple protein–protein interaction domains and are also known as proline-rich synapse-associated proteins (ProSAPs). Shank proteins are enriched in PSDs and stabilize the PSD-95/SAPAP/Shank/Homer complex . AdditioProSAP1/Shank2 gene result in an upregulation of glutamate receptors in certain brain regions, an increase in Shank3 at the synapse, and a decrease in dendritic spine morphology and synaptic transmission  synaptic mechanisms  which is a critical component of the PSD. At the PSD, SynGAP1 regulates synapse development and maintenance of proper synaptic function. It is known to interact with PSD-95 and colocalizes with excitatory NMDA receptor complexes . Discovery of more and more genes and genetic pathways are expanding the genetic landscape of ASD. It is interesting to note that these genes include chromatin modifiers, DNA binding proteins, ion channels, and neurotransmitter receptors  pathway.Mammalian target of rapamycin is a serine/threonine protein kinase involved in the regulation of cell proliferation, cell growth, cell survival, protein synthesis, and transcription. The mTOR signaling cascade plays a very important role in synapse protein synthesis and several studies have linked this pathway to ASD . Many ofCurrent efforts to identify the constellation of genes that confer the characteristic phenotypic manifestations within the autism spectrum have improved our understanding of this complex disorder. While modeling mutations in experimental animal model systems will highlight the underlying disruptions in conserved signaling pathways, the daunting task will still be to establish ASD-specific phenotypes at the molecular, cellular and neural circuit levels. The staggering number of genes already discovered in ASD holds the promise to translate the knowledge into designing new therapeutic interventions. The very interesting and equally challenging observation from the recent genetic studies has been a high degree of overlap of risk factors for various neurodevelopmental disorders, such as ASD, epilepsy, and schizophrenia. This pattern of overlap provides the feasibility to address which genes and genetic pathways intersect and specify the spatio-temporal sequence of events that occur within the developing human brain. The recent advent of comprehensive maps of spatio-temporal gene expression in the human brain  will greAlthough concerted efforts from studies of human clinical ASD populations and various ASD-related animal models have provided a better understanding of the genetic, molecular, and circuit level aberrations in ASD, several intriguing, yet significant questions still remain. For instance, how can the compound effects of genetics, epigenetics, and environment be consolidated in understanding ASD pathogenesis? What other events play a role in determining the appearance and trajectory of ASD symptoms? How do the majority of the genetic susceptibility loci in ASD affect synapse assembly, maintenance and functional modulation? Finally, how would the future treatments and interventions be designed and organized to accommodate the ever-changing genetic landscape of ASD?The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Resistant hypertension (RH) is defined as blood pressure (BP) that remains above the target of less than 140/90 mmHg in the general population and 130/80 mmHg in people with diabetes mellitus or chronic kidney disease (CKD) in spite of the use of at least three full-dose antihypertensive drugs including a diuretic or as BP that reaches the target by means of four or more drugs. In CKD, RH is a common condition due to a combination of factors including sodium retention, increased activity of the renin-angiotensin system, and enhanced activity of the sympathetic nervous system. Before defining the hypertensive patient as resistant it is mandatory to exclude the so-called “pseudoresistance.” This condition, which refers to the apparent failure to reach BP target in spite of an appropriate antihypertensive treatment, is mainly caused by white coat hypertension that is prevalent (30%) in CKD patients. Recently we have demonstrated that “true” RH represents an independent risk factor for renal and cardiovascular outcomes in CKD patients. Hypertension is defined “resistant” (RH) when blood pressure (BP) levels persist above the therapeutic target ), despite the use of at least three antihypertensive drugs at full dose, including the diuretic. Furthermore, according to the current definition, also hypertensive patients who reach BP target by means of four or more drugs are considered resistant .Although the exact prevalence is unknown, several observational studies suggest that RH is a common clinical problem in general population –8. In a Main causes of RH are reported in Before defining the hypertensive patient as resistant it is mandatory to exclude the so-called “pseudoresistance.” This condition refers to the “apparent” failure to reach BP target, in spite of an appropriate antihypertensive treatment. Among the causes of pseudoresistance , the mosThe second critical aspect for excluding pseudoresistance is the assessment of adherence to antihypertensive therapy. Lack of adherence is frequently encountered in clinical practice; indeed, nearly half of patients with hypertension withdraw therapy within the first year after diagnosis , and thaCKD is at the same time cause and complication of poorly controlled hypertension. The evaluation of RH in CKD patients is highly relevant for two main reasons. First, RH is common in CKD patients, and its prevalence increases with worsening of kidney damage  17]. Se. Se17]. Several surveys in CKD patients have demonstrated a high incidence of uncontrolled hypertension in clinical practice. Indeed the BP target is reached in only a small proportion (10–20%), both in nephrology and nonnephrology settings –24. However, uncontrolled hypertension is not equivalent of RH. This is particularly true in CKD patients in whom WCH is a common feature. Indeed, we have previously reported that WCH occurred in about 30% of patient with office BP ≥ 130/80 mmHg , and, asContemporaneous assessment of ABP monitoring, therefore, allowed to disclose a prevalence of “true” RH of 23–25% in CKD patients , 18, thaP = 0.004), and consequently the adherence to low-salt diet was poorer (P = 0.026)  .Therapeutic interventions aimed to inhibit the SRA and to reduce the ECV expansion frequently are insufficient at normalizing hypertension status in these patients, suggesting additional mechanisms in the pathophysiology of CKD-related hypertension. A series of experimental observations has allowed, in fact, generating new hypotheses such as the increase adrenergic activity, secondary hyperparathyroidism, and dysregulation of endothelial factors regulating the contractility of smooth muscle vessel . FinallyIn the population with essential hypertension, a relationship between RH and cardiovascular risk has been reported , 37. FurOf note, in pseudoresistant patients, ABP profiles, target organ damage  did not differ from normotensive patients, and their cardio-renal outcome was comparable to that of control patients . This reThe mechanisms underlying the different prognostic value of RH are not readily apparent; however, we can hypothesize that persistence of hypertension despite optimal antihypertensive treatment specifically identifies patients with more severe vascular damage. Diabetes, left ventricular hypertrophy, higher proteinuria, and, as mentioned, high salt intake, variables that we found independently associated with true resistance, are in fact all associated with endothelial dysfunction and arterial stiffness –45. In pThe multifactorial pathogenesis of hypertension in CKD imposes a multilevel treatment in these patients, even though to date there are no studies that assessed whether a particular combination of antihypertensive drugs is most advantageous to control hypertension in RH patients. The AASK study suggested that use of dihydropyridinic calcium-channel blocker (amlodipine) might not be adequate for use in CKD subjects, possibly because of increasing the GFR and thus inducing glomerular damage . More reIn CKD patients with RH, the pivotal intervention is certainly represented by the restriction of sodium intake. This dietary measure, however, is scarcely implemented as testified by the poor adherence (~20%) to low sodium diet even in patients regularly followed in nephrology clinics . This isThe critical role of salt retention in CKD-dependent hypertension precludes optimal control of BP during pharmacological treatment with antihypertensive agents, especially vasodilators  that are Ten recommendations to restrict sodium in your diet:(1) move the salt shaker away from the table;(2) cook pasta, rice, and cereals without salt ;(3) in cooking and at the table, increase the use of spices ;(4) abolish salt-containing condiments ;(5) look for the amount of sodium on food labels;(6) look for low-salt bread and fresh or plain frozen foods;(7) cut down frozen dinners, canned soups, packaged mixes, cured meat and fish ;(8) choose fresh rather than seasoned cheese;(9) rinse canned foods  to remove some sodium;(10) abolish salty snack foods .Improving compliance to a low-salt diet in hypertensive patients with CKD is helpful to make patient aware of the importance of low-salt intake to prevent progression of CV disease and CKD, to instruct patient on the correct way to collect 24-hour urine specimen, to monitor 24-hour urinary sodium excretion at each visit , to communicate to patient the daily salt intake estimated by 24-hour urinary sodium excretion , and to give specific recommendations on sodium restriction in diet listed in the following list.2) may respond to thiazide diuretics, those with more advanced CKD require the use of more potent loop diuretics administered at doses proportional to the reduced GFR [Generally diuretics are included in the definition of RH; however, in CKD patients the most crucial task of diuretic therapy is to properly select class and dose in relation to the level of kidney function. Indeed, if patients with mild renal impairment  due to the higher risk of hyperkalemia.A further diuretic agent successfully tested in RH patients is spironolactone based on the finding that plasma aldosterone levels are higher in RH that in those with controlled hypertension . Recentl2 were successfully treated with renal ablation with a significant systolic/diastolic BP reduction being evident after 1 month from intervention (−34/−12 mmHg) and persisting at one year (−33/−19 mmHg) [A novel therapeutic approach for RH is represented by catheter-based radiofrequency ablation of the renal sympathetic nerves, which was originally proposed in essential hypertension , 2. More19 mmHg) . However19 mmHg) . A trial19 mmHg)  but not RH is a common condition in CKD due to a combination of factors including sodium retention, increased activity of the renin-angiotensin system, and enhanced activity of the sympathetic nervous system. However, the higher prevalence of WCH in these patients imposes an out-of-office monitoring (ABPM or HBP) to distinguish between pseudoresistance and true RH. Therefore a more large use of ABPM in CKD patients is auspicable to attempt to limit the misclassification of hypertensive status in order to avoid unnecessary aggressive antihypertensive medication. To date the degree to which cardiovascular risk is reduced with treatment of resistant hypertension is unknown. Catheter-based radiofrequency ablation of the renal sympathetic nerves has been proposed, even though a greater implementation of a low-salt diet and a adequate use of the diuretic may be the first-choice therapeutic approach for controlling RH in CKD patients.


Human peripheral blood mononuclear cells (PBMCs) were differentiated into osteoclasts in the co-presence of dentin chips and biomaterials or dentin alone (control) for a period of 28 days. Osteoclast maturation was monitored on day 0 and 14 by light microscopy, and material-dependent changes in extracellular pH were assessed twice weekly. Mature osteoclasts were quantified using TRAP stainings on day 28 and their resorptive activity was determined on dentin (toluidin blue staining) and biomaterials . The analyzed biomaterials caused specific changes in the pH, which were correlated with osteoclast multinuclearity  and activity on biomaterials . Perossal led to a significant reduction of pH, nuclei per osteoclast and dentin resorption, whereas Tutogen bovine and Tutobone human strikingly increased all three parameters. Furthermore, natural biomaterials were resorbed more rapidly than synthetic biomaterials leading to differential relative resorption coefficients, which indicate whether bone substitutes lead to a balanced resorption or preferential resorption of either the biomaterial or the surrounding bone. Taken together, this study for the first time compares the effects of widely used biomaterials on osteoclast formation and resorbability in an unbiased approach that may now aid in improving the preclinical evaluation of bone substitute materials.Bioactive bone substitute materials are a valuable alternative to autologous bone transplantations in the repair of skeletal defects. However, clinical studies have reported varying success rates for many commonly used biomaterials. While osteoblasts have traditionally been regarded as key players mediating osseointegration, increasing evidence suggests that bone-resorbing osteoclasts are of crucial importance for the longevity of applied biomaterials. As no standardized data on the resorbability of biomaterials exists, we applied an  The repair of skeletal defects caused by trauma, infection or osteolytic bone metatases is one of the main clinical challenges of reconstructive surgery. For over 100 years the gold standard for repair has been the transplantation of autologous bone from the iliac crest, providing the advantages of both osteoconductive and osteoinductive action at the implanation site restitutio ad integrumAs an alternative, readily available and inexpensive allografts and xenografts have been developed, however they bear the risk of impaired integration into the surrounding bone tissue and, more importantly, may induce immunologic responses in the recipient with unpredictable consequences in vitro assay using human osteoclasts that can be co-cultivated with dentin (ivory), as a model of physiological bone, and any biomaterial of interest. Using this assay we were able to examine the differentiation of human osteoclasts, as well as osteoclast activity in the presence of polymethylmetacrylate (PMMA) or calcium phosphate cement (Biobon) We have previously reported an The first aim of this study was to evaluate the differentiation of human osteoclasts in the co-presence of dentin as a model of physiologic bone and several bone substitute biomaterials widely used in clinical practice . StartinThe second aim of this study was to determine potential effects of biomaterials on osteoclast activity. Therefore, the resorbed areas on dentin chips were quantified and normalized to the number of osteoclasts after 28 days of differentiation in the presence or absence (control) of the respective biomaterials. Interestingly, the vast majority of biomaterials  caused a 3- to 8.5-fold increase in dentin resorption per osteoclast relative to control p<0.05,  A +4 A. In the next step, resorption lacunae on biomaterials were quantified using scanning electron microscopy (SEM). Importantly, some of the materials revealed a rough, irregular or coarse surface at the beginning of the experiment , 0.48±0.13 (Tutobone human), 0.28±0.15 , 0.25±0.14 (Biobon) and 0.23±0.18 (Chronos)  B, respeSince the extracellular pH of culture medium has been reported to influence osteoclast formation and function restitutio ad integrum in patients with bone defects. However, despite the increasing use of synthetic biomaterials, contrary results regarding osseointegration have been reported and no applicable guidelines regarding the choice of appropriate bone substitutes exist in vitro.Given steadily improving surgical techniques, bone substitute materials are increasingly used to achieve Most biomaterials analyzed in this study led to significant changes in osteoclast formation, as assessed by morphological analyses of maturing mononuclear precursors and counting of TRAP positive multinucleated osteoclasts and nuclei at the end of the culture period. While several biomaterials caused an increase in the formation of mature osteoclasts, this effect was not observable in the case of Calciresorb, Tutogen bovine, Tutobone human, Biobon and Perossal. Tutogen bovine and Tutobone human led to significantly increased nuclei per osteoclast, whereas less nuclei per cell were observed in the case of Perossal. As the extracellular pH of culture medium has been reported to be a significant determinant of osteoclastogenesis and osteoclast function In the next step, potential differences in the resorbability of the respective materials were investigated using SEM. Among all biomaterials analyzed we found Lactosorb, Calciresorb, Cerasorb, Cerasorb M and Perossal were not assessable by SEM due to their coarse and irregular surface already present at the beginning of the experiment. Hence, it was virtually impossible to differentiate which changes of the material surface might have been due to osteoclast activity in these cases. Therefore, alternative methods like confocal laser scanning microscopy or infinite focus microscopy, as well as quantitative determination of biomaterial-degradation products in culture supernatants may have to be applied in order to quantify osteoclast resorption in vivoIn contrast to Tutogen bovine and Tutobone human, Biobon showed only a very limited resorbability. Again, using spearman rank coefficient analysis, we revealed a significant and positive correlation between the material-dependent extracellular pH changes and biomaterial resorption. Although Biobon was postulated to be a good alternative to autologous bone transplants, non-degraded biomaterial was still detectable one year after implantation As it seems unlikely that the differences in dentin and biomaterial resorption were exclusively caused by differential effects of the materials on osteoclastogenesis, they may at least in part be explained by modulating effects on osteoclast activity. In this context, it is important to state that proinflammatory cytokines, such as TNF-alpha and Interleukin 6, have been reported to act as potent inductors of osteoclast activity relative resorbability coefficient (RRC), which represents the quotient between resorption on biomaterial and resorption on dentin The quantification of osteoclastic resorption on biomaterials of interest often varies as no standardized method for differentiation of osteoclasts exists. Therefore, absolute resorption parameters differ between studies due to alternative protocols for osteoclast generation, cultivation and differentiation. To circumvent this problem and to make resorbability assays comparable, we have previously introduced the standardized parameter restitutio ad integrum. While several in vitro co-culture systems could be established imitating the bidirectional communication between osteoclasts and osteoblasts, there are no models for simultaneous analyses of osteoconductive and osteoinductive properties of bone substitutes Although the model applied in this study provides an important basis for the pre-clinical evaluations of bone substitutes regarding their effects on osteoclastogenesis and resorbability, we were not able to accurately determine total resorption on biomaterials and dentin chips. Due to the technical limitations of the applied methods it was only possible to determine the resorbed area, whereas the depth of resorption pits could not be assessed and requires measurement by infinite focus microscopy in future analyses in vivo and in patients. Moreover, our study defined relative resorption coefficients, which may now aid in improving preclinical assessment of bone substitute materials.In conclusion, this study compared the effects on osteoclast formation and resorbabilty of commonly used biomaterials using a human cell-based assay. Using this standardized and unbiased approach, we found a significant correlation between initial material-dependent changes in the pH of culture supernatants and osteoclast multinuclearity, as well as biomaterial resorption. Our assay is of potential clinical relevance, as the results are in line with previous reports on the resorbability of some of the materials 6 cells/cm2 in alpha-MEM  containing 0.22% sodium bicarbonate  containing the fraction of CD14+ precursors of human osteoclasts were isolated from buffy coats of healthy, consenting donors by density centrifugation using Ficoll Paque Plus  as described before pH = 6.9 , 10% FCSther day .Ten commonly used bone substitute materials were analyzed  N-N-Dimethylformamide and 30 mg Fast Red Violett LB Salt (Sigma) in 50 mL TRAP buffer (40 mM sodium-acetate and 10 mM sodium-tartrate in PBS) for 10 min. Cells with a positive staining for TRAP containing 3 or more nuclei were counted as osteoclasts. For quantification, the means of five representative fields of view in the pre-defined region of interest . After 28 days of incubation dentin and biomaterial chips were removed from the culture plates and subjected to the resorbability assay (see below). The cells were fixed with 3.7% formaldehyde  for 5 min and air-dried for 2 min. In order to determine the number of osteoclasts and the number of nuclei per osteoclast at the end of incubation, fixed cells were stained with TRAP staining solution containing 5 mg Naphthol AS-MX Phosphate (Sigma), 500 µL The biomaterial and dentin chips were washed with PBS to remove remaining cells. To visualize resorption lacunae, dentin chips were treated with 1% toluidin blue solution for the staining of collagen fibrils that are physiologically uncovered by the process of resorption (pit assay). The results were compared with a pit assay on day 0 of differentiation as an internal control, proving the complete absence of resorption lacunae on day 0. Unlike dentin, biomaterials do not necessarily contain collagen fibrils One aim of the study was to determine the clinically relevant question of which of the tested materials might be preferentially resorbed by osteoclasts in comparison to the surrounding bone tissue. Therefore, the relative resorption coefficient (RRC) was calculated for each material, which represents the ratio between resorption on biomaterial and resorption on dentin All cell culture experiments were done with groups of at least n = 5 and repeated at least three times with cells from two different donors. Statistical analysis was done using ANOVA with Bonferroni post-hoc test and Spearman correlation using IBM SPSS Statistics Version 20.0 . The probability of a type I error was set to 5% . Error bars represent standard deviation (SD).