Lysosomal storage diseases (LSDs) arise from monogenic deficiencies in lysosomal proteins and pathways and are characterized by a tissue-wide accumulation of a vast variety of macromolecules, normally specific to each genetic lesion. Strategies for treatment of LSDs commonly depend on reduction of the offending metabolite(s) by substrate depletion or enzyme replacement. However, at least 44 of the ~50 LSDs are currently recalcitrant to intervention. Murine models have provided significant insights into our understanding of many LSD mechanisms; however, these systems do not readily permit phenotypic screening of compound libraries, or the establishment of genetic or gene-environment interaction networks. Many of the genes causing LSDs are evolutionarily conserved, thus facilitating the application of models system to provide additional insight into LSDs. Here, we review the utility of yeast models of 3 LSDs: Batten disease, cystinosis, and Niemann-Pick type C disease. We will focus on the translation of research from yeast models into human patients suffering from these LSDs. We will also discuss the use of yeast models to investigate the penetrance of LSDs, such as Niemann-Pick type C disease, into more prevalent syndromes including viral infection and obesity. Neurodegenerative diseases represent one of the leading causes of disability in modern populations. Approximately five million Americans currently have Alzheimer’s disease, a number that is expected to be as high as 13.4 million by 2050 2Unfortunately, very few neurodegenerative conditions are treatable, and most worsen over time. Even monogenic neurodegenerative conditions, such as certain LSDs, where the biochemical and genetic etiology is very well defined, often remain tragically incurable, leaving palliative care as the only option to most patients. A novel and innovative rationale to treatment identification is clearly required. We propose that neuronal health is determined by very basic metabolic pathways, common to all cells and evolutionarily conserved from yeast to humans. Research models for polygenic disorders are hard to create and even harder to manipulate. By contrast, numerous spontaneous or induced models of rare monogenic disorders of high penetrance exist. These are frequently revealing with regard to disease mechanism and often establish therapeutic strategies for related polygenic pathologies (for example the LDL-Receptor and hypercholesterolemia). We have established a novel and innovative “exacerbate-reverse” approach to identify and exploit genetic loci that render cells sensitive to loss of genes defective in human monogenic diseases. We have successfully accomplished this strategy first in yeast and subsequently in mammalian cultured cell systems and murine models 464Saccharomyces cerevisiae) genome for the past 20 years or more has rendered what is now a relatively common application of global genome-wide screens to comprehensively place gene products and their associations in the context of whole cell physiology and pathophysiology The availability of a sequenced budding yeast  and genetic modifiers are identified as a consequence of exacerbation of the disease associated phenotype. This epistatic screen for genetic interactions is rendered disease specific by performing the screen under conditions that reflect or are known to impact the human disease state. The synthetic interactions, most often observed as a conditional inviability of the mutant combinations, are then targeted in the opposite direction to genetically or pharmacologically reverse disease phenotypes in a mammalian model of the human disease. It is this reversal stage of the process that leads to therapeutic interventions that are made relevant to human disease by only targeting conserved and “druggable” pathways, ideally with FDA approved agents.An approach we have termed “i.e., suppress) the disease severity of the LSD yeast model are identified. Accordingly, these reactions become potential drug targets whereby inhibitors would be predicted to reverse the disease phenotype in human cells. This strategy is limited to those disease models in which deletion of the ortholog has a clear phenotype in yeast.In contrast to “exacerbate-reverse”, a “rescue-reverse” approach can also be utilized. In this conceptually similar approach, gene deletions that directly rescue  arising from deficiencies in non-enzymatic lysosomal proteins, soluble lysosomal proteins or non-lysosomal proteins 414442Caenorhabditis elegans contains 58 genes orthologous to human genes associated with LSDs -ATPase 6470CTNS gene mutation, the disorder has three different clinical presentations: infantile nephropathic, juvenile nephropathic and ocular non-nephropathic 65737576Cystinosis is an autosomal recessive lysosomal storage disorder with the clinical hallmark of insoluble cysteine accumulation in the lysosome 65666568CTNS gene product is highly conserved with the Ers1p protein of S. cerevisiae  ERS1 is a functional ortholog based on complementation of hygromycin B (hygB) sensitivity of ers1Δ deletion mutant strains by heterologous expression of human CTNS CTNS mutants in mammalian cells, loss of ERS1 does not affect yeast viability and no accumulation of cystine is observed Whole animal and cultured mammalian cell models have been developed and utilized to further understand the pathogenic mechanisms in cystinosis 647981ers1Δ strains implicated sulfur homeostasis as aberrant, which was consistent with the gene expression profile of cystinotic cells from cystinosis patients. Additionally, energy production, oxidative stress, calcium and potassium transport and stress response were also awry in this mutant model MEH1, a gene critical for the process of microautophagy and the function of amino acid permease Gap1p that mediates cysteine uptake, suppressed hygB sensitivity in the ers1Δ yeast model 84MEH1 also was identified in an interaction with GTR1, a small Ras-related GTPase involved in macroautophagy MEH1 and GTR1 in yeast, as well as the human orthologs in mammalian cells, may provide more insight into the molecular mechanism of cystinosis. It would also be informative to identify gene deletions that suppress hygB sensitivity of ers1Δ, an experiment that can be easily conducted in yeast via a Synthetic Genetic Array (SGA) analysis wherein the ers1Δ strain is crossed with the deletion library to generate haploid double deletion strains. As hygB sensitivity in yeast is a marker for disease severity in humans, this analysis would provide a list of candidate genes and pathways that modify disease severity. More broadly, investigations of these modifiers may identify the compensatory mechanism(s) that prevent the accumulation of cystine in yeast and thus create a more powerful yeast model of cystinosis with a similar phenotype to humans.Given this conservation, the yeast model has provided significant insight into the molecular mechanisms underlying cystinosis particularly with respect to pathways that may modify cystine accumulation in patients. A comparative transcriptomic analysis of CLN3 locus being the most common cause of juvenile NCL, also known as Batten disease. 6392CLN3 gene encodes a multipass transmembrane protein that localizes to the lysosomal, endosomal and autophagosomal compartments 9496Batten disease encompasses a group of fatal, pediatric, autosomal recessive lysosomal storage disorders that are the most common form of neuronal ceroid lipofuscinosis (NCL) 8788CLN3, CLN8, CTSD, DNAJC5, and PPT1) are conserved in yeast (Table 1). Although large animal  and murine models have been developed to study the mechanism of juvenile NCL, the evolutionary conservation of CLN3, the most common cause of Batten disease, allows the use of lower eukaryotic genetic models to study the disorder 98S. cerevisiae ortholog of human CLN3 sharing 37% amino acid identity BTN1 deletion strains exhibited increased plasma membrane ATPase activity, acidified growth media and resistance to D-(-)-threo-2-amino-1-[p-nitrophenyl]-1, 3-propanediol (ANP) BTN1 is apparently not involved in degradation of mitochondrial ATPase subunit c, however expression of human CLN3, complements the ANP resistance of btn1Δ yeast, indicating that BTN1 and CLN3 are functionally conserved 101100btn1Δ strains 103btn1Δ yeast strains revealed increased resistance to menadione-generated oxidative stress via impaired nitric oxide synthesis and decreased levels of arginine The obscure etiology of Batten disease warrants a new approach both to mechanism and treatment. Five of the 14 NCL genes  disease , is a complex lipid lysosomal storage disorder, typified by accumulation of low-density lipoprotein-derived unesterified cholesterol, sphingomyelin, glucosylceramide and sphingosine 6113114115S. cerevisiae) as a genetically tractable and valid model of NP-C disease by several criteria NCR14NCR1 encode proteins with 33% sequence identity. This identity persists to specific homology domains such as the sterol sensing domain (SSD) and the Patched homology domains. In general, residues key to the activity of this protein, as defined by the occurrence of specific disease mutations in NP-C patients are also conserved. Moreover both yeast and mammalian NPC1 proteins localize to limiting membranes of the lysosomal (vacuolar)/endosomal system 910btn1Δ and ers1Δ yeast LSD models, yeast strains deficient in NCR1 do not exhibit a striking growth defect, although Vilaca and colleagues have implicated the NCR1 mediated pathway in chronological lifespan (CLS), a measurement of non-replicative aging in yeast. Reduced CLS can be rescued with deletion of genes mediating sphingolipid metabolism further supporting a role of NCR1 in sphingolipid homeostasis NCR1-deficient yeast cells do not display any obvious defect in sterol metabolism, although membrane microdomains and microautophagy are aberrant during quiescence 49122124125NCR1 prompted the hypothesis that the ancestral function of NPC1 was in recycling sphingolipids ncr1Δ yeast variant 123. The lack of a compelling null phenotype in yeast NCR1 deletion strains, combined with our findings on conservation of these pathways between yeast and mammals provoked a series of screens for modifiers of NCR1 in yeast ncr1Δ strain only in the context of sterol auxotrophy (induced by anaerobiosis) ncr1Δ during anaerobic growth. This observation prompted the hypothesis that histone acetylase activity was reduced (and by inference histone deacetylase (HDAC) activity was increased) in human NP-C disease 5127128The NP-C proteins have been proposed as cholesterol transporters, in part because they both possess lipid-binding domains. However, the affinity of cholesterol for NPC1 is orders of magnitude lower than for NPC2, thus cholesterol binding to NPC1 may act catalytically, as opposed to bulk transfer or solubilisation 1177120nihNpc1 variant, defined by a null allele of Npc1, and nmf164Npc1 ; both animal lines develop a chronic, debilitating neurological disease and die between 9-12 and 13-16 weeks of age, respectively . Using nmf164Npc1 mice, we conducted a study to assess the efficiency of Vorinostat as potential therapeutic for NP-C disease. We determined that Vorinostat normalized transcriptional regulation of eight key genes critical for cholesterol homeostasis in the liver, thereby improving liver function and pathology nihNpc1 and nmf164Npc1 mice. This in vivo result is consistent with Vorinostat-mediated reduction in filipin fluorescence in 52 of 60 different NPC1 mutations in cultured cells Our exacerbate-reverse approach identified Vorinostat as a candidate therapy in 2011 and Vorinostat was approved to treat cutaneous T-cell lymphoma in 2006. Hence a Vorinostat clinical trial was fast-tracked without proven therapeutic efficacy in an animal model of NP-C disease since there was no approved therapy to treat this fatal, pediatric disease. We subsequently tested two distinct murine models of NPC1 deficiency for their phenotypic response to HDAC inhibitors: The tau and amyloid-β peptides. Here we briefly review the importance of investigating NP-C disease to further understand AIDS, infectious haemorrhagic fever, and obesity. In particular we will describe the application of the aforementioned genetic strategies to establish novel approaches to therapeutic interventions for these high impact diseases.As mentioned previously, aberrant lysosomal function and cellular degradation has implications for all cells and thus contributes significantly to both health and disease 14, togaviruses such as Chikungunya, and Flaviviruses  131Among the conserved LSD-causing genes in yeast, six genes were also associated with viral assembly, entry, and replication (Table 1) 131132133134135Filoviridae 134135136138140ncr1Δ yeast model of NP-C disease.Ebola viruses (EBOV) cause infectious haemorrhagic fever and are classified in the family of ncr1Δyeast model of NP-C disease is relatively straightforward given that different proteins encoding for HIV-1  have been studied using yeast models  in humans. HIV-1 is a complex retrovirus that is dependent on cholesterol as a cellular platform for replication 144145147148We hypothesize a pivotal role of the NP-C pathway, particularly the NPC1 protein, as a nexus in lysosomal lipid metabolism that prevents the lipid toxicity that underlies many metabolic disturbances such as obesity and diabetes. A convincing genome wide association study (GWAS; multiple populations, multiple polymorphisms, both genders; 11121315161718156S. cerevisae is a simple eukaryotic model that generates biochemical and genetic output that can readily be translated into more complex eukaryotes. Hence, screening for monogenically determined disease modifiers using a yeast LSD model and translating it into higher eukaryotes saves time and cost in identifying the therapeutic drug target. Although there are some limitations encountered when using a yeast model of LSDs, we demonstrated these can be overcome by using experimental conditions that mimic the processes defective in human patients. The “exacerbate-reverse” approach described here has great potential to contribute to our understanding of LSDs with specific emphasis on drug discovery. Our discovery of Vorinostat as a candidate therapy now in clinical trial has established this strategy as a promising pipeline to LSD treatments btn1Δ yeast model of Batten disease and ANP resistance to identify modifiers of Batten disease and to the ers1Δ yeast model of cystinosis via a “rescue-reverse” of hygB sensitivity as potential drug targets to treat cystinosis. Apart from discovery of drug targets to treat LSDs, yeast is a powerful tool to study virus infection and pathogenesis. The molecular mechanism of virus-host interactions and the characterization of the host functions can be easily elucidated using the genetic tools available. Remarkably, interaction of each individual viral-encoding protein can easily be studied in yeast via heterologous expression of each protein in yeast, which can then be integrated into powerful genome-wide analyses of various mutant libraries. These analyses would direct the discovery of antiviral treatments by identifying genes that prevent virus entry or replication. In summary, genome-wide analyses that exacerbate or ameliorate yeast growth phenotypes caused by yeast gene mutations or viral proteins may prove advantageous to identify genes and pathways that modify human genetic diseases and infection pathogenesis.In summary, the rapid and continuing advancement of genomic and proteomic research in yeast is a key advantage to identifying genetic modifiers as therapeutic drug targets.


Adipocytes are master regulators of energy homeostasis. Although the contributions of classical brown and white adipose tissue  to glucose and fatty acid metabolism are well characterized, the metabolic role of adipocytes in bone marrow remains largely unclear. Here, we quantify bone fatty acid metabolism and its contribution to systemic nutrient handling in mice. Whereas in parts of the skeleton the specific amount of nutrients taken-up from the circulation was lower than in other metabolically active tissues such as BAT or liver, the overall contribution of the skeleton as a whole organ was remarkable, placing it among the top organs involved in systemic glucose as well as fatty acid clearance. We show that there are considerable site-specific variations in bone marrow fatty acid composition throughout the skeleton and that, especially in the tibia, marrow fatty acid profiles resemble classical BAT and WAT. Using a mouse model lacking lipoprotein lipase (LPL), a master regulator of plasma lipid turnover specifically in adipocytes, we show that impaired fatty acid flux leads to reduced amounts of dietary essential fatty acids while there was a profound increase in de novo produced fatty acids in both bone marrow and cortical bone. Notably, these changes in fatty acid profiles were not associated with any gross skeletal phenotype. These results identify LPL as an important regulator of fatty acid transport to skeletal compartments and demonstrate an intricate functional link between systemic and skeletal fatty acid and glucose metabolism. N-acyl amides have been implicated in bone remodeling through both stimulation of bone formation as well as inhibition of bone resorption -linoleic acid, 12 kBq/g body weight) and palmitic acid  or glucose  and palmitic acid ]-linoleic acid, 12 kBq/g body weight) were performed as described previously [All animal experiments were approved by the Animal Welfare Officers of University Medical Center Hamburg-Eppendorf  and Behörde für Gesundheit und Verbraucherschutz Hamburg . C57BL/6J mice were purchased from Jackson Labs  and bred and housed in the animal facility of the University Medical Center Hamburg-Eppendorf at 22 °C with ad libitum access to water and standard laboratory chow diet . Animals carrying floxed alleles of alidated . Standareviously . The conPlasma triglycerides and cholesterol were determined using commercial kits  that were adapted to microtiter plates. For fast performance liquid chromatography (FPLC) pooled plasma was separated using S6-superose columns  and lipid levels were analyzed in each fraction as described above. Bone turnover markers were measured according to the manufacturers’ instructions: Alkaline phosphatase was determined using the NPP method , OPG and RANKL were determined by ELISA , OCN was analyzed by an immunoradiometric assay  and DPD/creatinine were determined by ELISA . Blood glucose levels were measured using AccuCheck Aviva sticks (Roche) as described previously .® (Invitrogen) using a TissueLyser . Total RNA was isolated using NucleoSpin RNA II kit . Complementary DNA was synthesized using SuperScript® III Reverse Transcriptase (Invitrogen). Quantitative real-time PCR reactions were performed on a 7900HT sequence detection system  using TaqMan Assay-on-Demand primer sets supplied by Applied Biosystems.Tissues were immediately placed into RNAlater , incubated over night and then disrupted in TRIzolHigh-resolution µCT imaging of the right femur and tibia was performed at a resolution of 10 µm per voxel using a Scanco µCT 40  according to the guidelines of American Society for Bone and Mineral Research ,52. The Tissue lipid extracts by the Folch method and total tissue fatty acid profiling by gas chromatography were performed as described in detail recently using 50 µL of solvent per mg of tissue . Concentt-test were used for comparison of groups or genotypes. p < 0.05 was considered significant. Fatty acid profiles were corrected for multiple testing and significance adjusted accordingly.One-way ANOVA or Student’s Skeletal compartments significantly contribute to whole body clearance of glucose and fatty acids and display location-specific differences in fatty acid composition. Adipocyte lipoprotein lipase, which is a gatekeeper for the uptake of lipoprotein-derived fatty acids, profoundly affects the uptake and composition of marrow adipocytes in a location-specific manner in the skeleton.


Here we sought to advance this model in regard to lethality for future countermeasure efficacy testing against more recent ZIKV strains from the Asian lineage, preferably the American sublineage. We first infected IFNAR−/− mice subcutaneously with the contemporary ZIKV-Paraiba strain resulting in predominantly neurological disease with ~50% lethality. Infection with ZIKV-Paraiba by different routes established a uniformly lethal model only in young mice (4-week old) upon intraperitoneal infection. However, intraperitoneal inoculation of ZIKV-French Polynesia resulted in uniform lethality in older IFNAR−/− mice (10–12-weeks old). In conclusion, we have established uniformly lethal mouse disease models for efficacy testing of antivirals and vaccines against recent ZIKV strains representing the Asian lineage.The common small animal disease models for Zika virus (ZIKV) are mice lacking the interferon responses, but infection of interferon receptor α/β knock out (IFNAR Aedes aegypti mosquitos and propagates in vertebrate hosts  is a human pathogen that was first isolated in 1947 from a sentinel rhesus monkey in the Zika forest of Uganda Dick, . The virs Hayes, . In humas Hayes, . Symptoms Hayes, . For mans Hayes, . Additios Hayes, , and thes Hayes, . ConsideFlavivirus genus. It shares many structural similarities with other pathogenic flaviviruses, such as Dengue virus (DENV), Japanese encephalitis virus (JEV), and West Nile virus (WNV)  mouse model as these mice, despite being immunocompromised, can still develop protective immune responses  and ZIKV-French Polynesia in older (10–12-weeks old) IFNAR−/− mice. Thus, we were able to establish age-dependent uniformly lethal IFNAR−/− mouse models suitable for countermeasure efficacy testing against recent ZIKV strains.The most amenable small animal models for ZIKV infection are mice lacking interferon responses recapitulating certain features of human disease like meningoencephalitis and fetal brain malformations  and conducted in compliance with all necessary guidelines and regulations. Our research facility is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC) and has an approved Office of Laboratory Animal Welfare (OLAW) Assurance number (#A4149-01). All procedures were conducted by trained personnel under the supervision of veterinarians, and all invasive clinical procedures were performed while animals were anesthetized. Humane endpoint criteria, as specified by the IACUC approved scoring parameters, were used to determine when animals should be humanely euthanized. ZIKV is classified as biosafety level 2 (BSL2) pathogen and all work with the ZIKV isolates was approved by the Institutional Biosafety Committee (IBC) under BSL2 conditions.KX280026.1)  and incubated overnight at 37°C. The next day, 10-fold serial dilutions of the ZIKV stock were prepared in complete DMEM. Cells were infected with 125 ul of each dilution to be tested in duplicate at 37°C for 1 h. Virus was aspirated and wells were washed 1X with PBS. Cells were overlayed with 1 ml of 1.5% carboxymethylcellulose (CMC) (Sigma) dissolved in MEM and incubated at 37°C for 4–5 days. For fixation, 1 ml of 10% formalin was added directly on top of the CMC and plates were incubated at room temperature overnight. Wells were washed 3–4 times with water to remove the CMC and 1% crystal violet was added to each well for 15–30 min at room temperature to visualize plaques. Wells were washed with water, plaques were counted and titer was calculated.VeroE6 cells were plated at 2 × 10Total RNA was extracted from tissues using the RNeasy Mini Kit (Qiagen) according to manufacturer's directions. Total RNA was extracted from whole blood using the QIAamp Viral RNA Mini Kit (Qiagen) according to manufacturer's directions. ZIKV-specific RNA was detected using the primers and probe set described by Faye and colleagues  were calculated using the Reed-Muench method  and the median tissue culture infectious doses (TCID−/−) mice ranging from 4–12 weeks of age were inoculated with the indicated doses of ZIKV (diluted in sterile DMEM without supplements) by either subcutaneous (between the shoulder blades) or intraperitoneal injection in a total volume of 100 μl. Additionally, footpad (left rear foot) inoculation was performed with a 50 ul inoculum per mouse. The animals were monitored for signs of illness and weighed daily. On necropsy days, mice were anesthetized, bled, euthanized and tissue samples were taken and stored at −80°C until titration. Surviving mice were euthanized 28 days after infection.Female and male C57BL/6 interferon receptor α/β knock out . Tissue samples were evaluated in detail and the following scoring system was applied: 0 = no lesions; 1 = small number of necrotic cells; 2 = moderate necrosis; 3 = significant necrosis; 4 = coalescing necrosis; 5 = diffuse necrosis. Detection of ZIKV RNA was performed using the RNAscope 2.5 VS assay (Advanced Cell Diagnostics Inc.) on the Ventana Discovery ULTRA as previously described  was performed . All survival curves were compared with the Log-rank (Mantel Cox) survival test. Body weight curves were analyzed using parametric in vitro growth kinetics of three different ZIKV strains representing the African and Asian lineage as well as the American sublineage  to more closely mimic natural infection and observed the mice daily for signs of disease including changes in body weight. On days 3 and 8 after infection, a subset of the animals was euthanized to collect blood and tissue samples for virus load determination and pathology. Starting on days 6 and 7 post-infection, the animals lost weight independent of the dose of ZIKV  demonstrated no lesions that could be attributed to ZIKV infection (data not shown). However, by day 8 after infection, animals in both groups  developed hepatic . We infected groups (mixed gender in each group) of 6 mice with 105 PFU of ZIKV-Paraiba by the footpad, s.c. or i.p. route and documented disease progression. Independent of the route of infection, very few of the older mice demonstrated significant weight loss or other signs of disease  as 1 PFU in young mice  with two additional ZIKV strains representing the African and Asian lineages   resulted in similar signs of disease and neuropathological brain changes  as described previously for A129 mice at the same age infected with an African ZIKV strain by the same route  independent of the route of inoculation (Figure 5 PFU i.p. resulted in uniform lethality, whereas all other groups contained a subset of animals that recovered from infection (Figure For the establishment of a challenge model for vaccine efficacy testing, older mice needed to be evaluated as developing immunity upon vaccination requires time. Other groups have used AG129 mice deficient of type I and II interferon responses to achieve uniform lethality in control/placebo groups despite their limitations in responding to vaccination (Richner et al., n Figure . Therefon Figure  and, the−/− mouse models utilizing two distinct ZIKV strains form the Asian lineage that are suitable for efficacy testing of treatment and vaccine approaches.In summary, we were able to establish uniformly lethal IFNARAM and HF: designed the studies; AM, JE, JC, KM, NA, SC, and CM: performed the experiments; RR and DPS: processed and analyzed the pathology samples. AM, DS, SW, SB, and HF: analyzed the data; AM and HF: wrote the manuscript. All authors approved the manuscript.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.


Focal adhesion kinase (FAK) plays a primary role in regulating the activity of many signaling molecules. Increased FAK expression hasbeen associated in a series of cellular processes like cell migration and survival. FAK inhibition by an anti cancer agent is critical.Therefore, it is of interest to identify, modify, design, improve and develop molecules to inhibit FAK. Solanesol is known to haveinhibitory activity towards FAK. However, the molecular principles of its binding with FAK is unknown. Solanesol is a highly flexibleligand (25 rotatable bonds). Hence, ligand-protein docking was completed using AutoDock with a modified contact based scoringfunction. The FAK-solanesol complex model was further energy minimized and simulated in GROMOS96 (53a6) force field followedby post simulation analysis such as Root mean square deviation (RMSD), root mean square fluctuations (RMSF) and solvent accessiblesurface area (SASA) calculations to explain solanesol-FAK binding. Cancer has affected more than 15 million people across the globeand it is expected to affect 25 million people in the next 20 years. The inhAngiogenesis is the process in which new blood vessels originatefrom the existing blood vessel system . InnumerFocal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase(PTK2) that plays a primary role in growth factor mediatedsignaling and mediates significant role in cell migration,proliferation, and angiogenesis . SeveralNatural products have a huge reputation as promising anticanceragents and in the modern era, naturally derived productsare frequently accepted in therapy among the masses . A lotoSolanesol is a non-cyclic polyisoprenoid alcohol (composed ofnine isoprenoid unit) present mainly in solanaceous crops liketobacco, tomato, potato, eggplant, and pepper plants .SolanesThe present research involves establishing of solanesol as a focaladhesion kinase (FAK) inhibitor by applications of computationalin silico methods. Though, solanesol used as a bioactive agent inindustries for decades, due to its highly flexible nature, there isno successful in silico protein binding and simulation dataavailable online till date. We also proposed a method of bindingof the highly flexible compound to protein targets using anenhanced contact based scoring method. This method scores theresidues rather than the conformations. The higher scoredresidues were then used for more "focused" docking on thoseresidue regions.The crystallographic co-ordinates for Focal adhesion kinase (PDBID: 4Q9S)  were retSolanesol exist in both cis and trans states, for this experiment weconsidered only trans is found in natural sources . ThestrAll the molecular docking studies of Solanesol to FAK wereperformed using Autodock 4.2 . AutodocSolanesol is a 45-carbon chain with 26 rotatable bonds. As it isextremely flexible it is hard to determine the bind mode of it with the protein. The commonly used protocol for determination ofbinding pocket is "Blind docking", which was initially developedfor to determining peptide docking with protein . In thisFor calculating the possible area of interaction or binding site of ahighly flexible ligand we enhanced blind docking using LigandContact Based Scoring function for Residues (LCBSR). This is anatomic contact/clash based scoring method, in which theresidues are scored based on the higher favorable interactionsand probability of formation of a hydrogen bond. As it doesn'tdepend on the clustering or solely on binding energy, itstatistically enhances the probability of finding the possiblebinding site.It can be represented as,LBCSR = log(NCo - NCl) - log(NH/CCl) (1)Where, ligand contact based scoring of residue (LCBSR) iscalculated using "Number of Contacts" (NCo) which is thenumber of occurrences where atoms of residue (r) are in contactwith any atom of the ligand in all the conformations. "Number ofClashes" (NCl) is the number of occurrences when atoms ofresidue (r) are in clashes or unfavorably overlapped with anyatom of the ligand in all the conformations. "Number ofHydrogen Bonds" (NH) is the number of occurrences whenatoms of residue (r) are forming a hydrogen bond with any atomof the ligand in all the conformations. "Number of Clashes" (CCl)is the number of conformers where residue (r) is in anunfavorable overlap with any atom of the ligand.The "Contact" here is defined as the instance when the differencebetween the distance of two atoms and the sum of their van derWaal radii is 0.4 A or more or, in other words, the distance isgreater than the sum of van der Waal radii (Eq. 1) of two atoms.Whereas "Clash" is defined as the condition where the van derWaal radii of two atoms unfavourably overlaps each other andthe distance is lower than the sum of radii (Eq. 2) of the twoatoms.This can be represented as:∑rVDW - Dij ≥ -0.4 (2)∑rVDW - Dij ≥ 1.0 (3)Where, ∑rVDW is the sum of van der Waal radii of interactingatoms of ligand (i) and residue (j) and Dij is the distance betweeninteracting atoms of ligand (i) and residue (j). A Higher value ofLCBSR of any residue implies the residue may be a part of thebinding pocket for the ligand and, if it is capable, it may alsoform a hydrogen bond with the ligand. Lesser score implies a lower interaction or high chances of unfavorable clashes or lowchances of forming a hydrogen bond.Solanesol has 25 rotatable bonds, which makes very difficult todock directly to the protein structure by conventional method.Although, there are various methods for prediction of bindingsite in FAK, the flexible nature of solanesol as well as the size ofthe compound makes it difficult to fit the active site. For thepurpose of predicting the actual binding site, blind dockingmethod can be used. Blind docking method was introduced forthe purpose of docking peptide molecules to the protein moleculebut is a tested method for binding of small molecules whenbinding site is unknown . SolanesBinding site for Solanesol was considered using residues with ahigher LCBSR score. This binding site was then used for refineddocking using Autodock. The experiment was done twice with(1) relaxed parameters, GA maximum energy evaluations 2.5 x106, for 200 GA runs (2) exhaustive parameters, GA maximumenergy evaluations 3.5 x 107, for 200 GA runs.All conformations were rescored using DSX, Drug ScoreeXtended , knowledMolecular dynamics simulations for FAK protein as well asSolanesol bound FAK were performed using the GROMACS 4.6  softwareFor electrostatic calculations, Particle Mesh Ewald (PME) method wThe root mean square deviation (RMSD) and root mean squarefluctuations (RMSF) of FAK backbone were calculated using "g_rms" and "g_rmsf" utility commands, respectively. Aspherical probe of radius 1.4 Å across the protein surface wasused for calculating solvent-accessible surface area (SASA) by"g_sasa" tool of Gromacs. Hydrogen bonds between Solanesoland FAK were calculated using "g_hbond" tool with protondonor and acceptor distance ≤ 3.5 Å and the angle betweenacceptor-donor-hydrogen ≤ 30.0 degrees.The molecular mechanics Poisson-Boltzmann surface area(MMPBSA)  approachΔΔGBE=ΔGComplex- (ΔGReceptor + ΔGLigand) (4)ΔG = ΔEMM + ΔGSol - TΔS (5)ΔEMM = ΔEint + ΔEele+ ΔEvdw (6)ΔGSol = ΔGPB + ΔGSA (7)ΔEMM, ΔGSol and TΔS represent the molecular mechanicscomponent in the gas phase, stabilization energy due tosalvation, and a vibrational entropy term, respectively. ΔEMM isthe summation of ΔEint, ΔEcol, and ΔEvdw, which are the internal,coulomb, and van der Waals interaction terms, respectively. ΔGsolis the salvation energy and it is divided into an electrostaticsalvation free energy (GPB) and a non-polar salvation free energy(GSA).Solanesol shows very small cluster with insignificant bindingaffinity towards FAK when docked "blindly" . Though,From all the generated conformations of Solanesol, which werehaving binding energy lower than -4.0 kcal/mol, all the favorableand unfavorable overlaps of the atoms were calculated using"FindClash" tool of UCSF Chimera. Chimera tool, "FindHbond"was also used to find the hydrogen bonds between the ligandand residue atoms. The default values were kept for all thecalculation in Chimera.An in-house python script was used to calculate the number of"Clashes", "Contacts" and "Hbonds" between all theconformations and residues as well as individual scores. Thescores from all the three experiments were added to give a finalscore for each residue .Based on the LBCSR score calculated for all three experiments,the scores for all the residues were plotted as graph  aswell All the residues with more than LBCSR score was considered forthe active site prediction. A total of 77 residues were found to beabove and incidentally which also forms the ATP binding siteand the catalytic loop (546-551) and formed between the N and Clobe.The centre of geometry  of these 77residues was considered for the centre of the binding pocket ofSolanesol. A grid of size 60 x 62 x 68 was considered to exactly fitall the 77 residues. Autodock4.2 was again used for docking ofSolanesol with FAK with this grid setting for two more times,first time with default setting for 200 conformations and laterdocked with exhaustive setting for the same number ofconformations. All 400 conformations were rescored using DSXonline server with CSD settings .The hydroxyl end of Solanesol binds to the binding site of ATPand interacts with Ile428, Val436, Ala452, Lys454 and Leu501. These rThese 12 hydrophobic interactions of 11 residues with Solanesolstabilize the ligand at the middle of N and C lobe of FAK. As theligand share interacting residues with both the side it may be actas a better inhibitor for FAK. This structure was used formolecular dynamics studies.For the measuring the stability, the RMS deviation in thebackbone of the Solanesol bound FAK and only FAK beenmeasured and plotted in accordance to time . After aComparison of the RMSD of both the trajectories from theminimized structures shows, Solanesol bound FAK backboneshow more stability than that of independent FAK backbone.RMSF (Root mean Square Fluctuation) of Solanesol bond FAKexhibits less fluctuation at the places where Solanesol is bond.Total solvent accessible surface area (SASA) was checked byg_sas tool of Gromacs for analyzing the change in surface areawith respect to time. It show a quite negative correlation with theRMSD suggesting the better binding of the ligand leads to alower surface area of the protein thus closing the active site forany further contact. It remains stable for the wider part of 6ns -18ns range after which the backbone gets stabilized and may alsoaffect the surface area.Binding site residues for Solanesol were selected by taking 3.5Aradius from Solanesol. Molecular mechanics Poisson-Boltzmannsurface area (MM-PBSA) was calculated for the last 5 ns with 20ps steps for the binding site residues versus Solanesol usingg_mmpbsa tool. Per residue analysis of the result was done andplotted using the script provided. This analysis suggests that,Ile428, Gly431, Val436, Val484, Met499, Cys502 and Leu553interacts most favorably with the ligand . InteresWe report the binding of Solanesol with FAK using aGROMOS96 (53a6) force-field simulated docked model ofSolanesol-FAK complex. The binding of solanesol at the ATPbinding site to inhibit the phosphorylation of FAK is explained.Data help to understand binding of flexible compounds withFAK for potential inhibition.


Strikingly, insertion of a human chromosome into mouse revealed that promoter-associated CGIs are refractory to DNA methylation regardless of host species, demonstrating that DNA sequence plays a central role in specifying the hypomethylated state through evolutionarily conserved mechanisms. In contrast, elements distal to gene promoters exhibited more variable methylation between host species, uncovering a widespread dependence on nucleotide frequency and occupancy of DNA-binding transcription factors in shaping the DNA methylation landscape away from gene promoters. This was exemplified by young CpG rich lineage-restricted repeat sequences that evaded DNA methylation in the absence of co-evolved mechanisms targeting methylation to these sequences, and species specific DNA binding events that protected against DNA methylation in CpG poor regions. Finally, transplantation of mouse chromosomal fragments into the evolutionarily distant zebrafish uncovered the existence of a mechanistically conserved and DNA-encoded logic which shapes CGI formation across vertebrate species.DNA methylation is a repressive epigenetic modification that covers vertebrate genomes. Regions known as CpG islands (CGIs), which are refractory to DNA methylation, are often associated with gene promoters and play central roles in gene regulation. Yet how CGIs in their normal genomic context evade the DNA methylation machinery and whether these mechanisms are evolutionarily conserved remains enigmatic. To address these fundamental questions we exploited a transchromosomic animal model and genomic approaches to understand how the hypomethylated state is formed  DNA methylation on CpG dinucleotides in vertebrate genomes is associated with transcriptional repression ,2 and isDespite our ever-increasing knowledge of DNA methylation landscapes in diverse vertebrate genomes, the principles and mechanisms that specify these patterns and protect CGIs and distal regulatory elements from DNA methylation is underexplored, particularly with respect to how these mechanisms function in their normal genomic context on chromosomes and during animal development. This is due to the fact that most of our information about these processes has relied on interrogating CGIs and their methylation states by inserting short DNA sequences  into engineered acceptor sites in cell culture systems ,21–24. TTo explore the mechanisms that shape CGI methylation state in a genomic context and to directly test whether these properties are evolutionarily conserved in vertebrates we have exploited a transchromosomic mouse animal model (Tc1)  in whichThe Tc1 mouse line was maintained as previously described . Tc1 micFour mouse BACs were engineered to contain a GFP reporter driven by the eF1alpha promoter, and an inverted Tol2 transposition cassette (iTol2) flanking an ampicillin resistance gene  by BAC rBioCAP-sequencing was performed as described previously  in Tc1 mTc1 BioCAP-sequencing reads were aligned to a composite genome containing mouse chromosomes and human chromosome 21 using bowtie . HMRs weCpG density and GC content of individual HMR regions was calculated based on the underlying DNA sequence for HMRs that were shared between human and Tc1 mouse or were species-specific. A matched background control was generated by randomly shifting HMR coordinates to another position on chromosome 21. CpG density and GC content was plotted as frequency distribution plots for liver and testis tissue.−9 per year  and dividing this number by the estimated mutation rate for mammalian species, which is 2.2 × 10per year ,36. The Transcription factor binding sites were identified using the MACS1.4 peak caller with the same settings as were used to identify HMRs above . Shared Gene expression data  was used to compare the expression of genes with an HMR overlapping their promoter (TSS ± 500bp) . Genes wHeatmaps and metaplots were generated using Homer annotatePeaks and plotted in R. Venn diagrams were plotted in R using the package VennDiagram. Boxplots were plotted in R using the package boxplot. Scatterplots were generated in R.Arabidopsis thaliana BAC DNA (F24B22 and F19K16 respectively) was spiked into the three conversion reactions to control for bisulfite conversion . PCR-amplified DNA was pooled for each replicate sample and Illumina sequencing libraries were prepared using the NEBNext DNA Library Prep Master Mix Set for Illumina (NEB). Indexed libraries were combined and sequenced on a MiSeq machine (Illumina). Sequencing data was analysed using the BisMark software from Babraham Bioinformatics (http://www.bioinformatics.babraham.ac.uk/projects/bismark/) (Bisulfite conversion of 250 ng human and two replicates of Tc1 mouse liver genomic DNA was performed using the EZ DNA Methylation-Gold Kit (Zymo Research). 10 picogram (pg) unmethylated and 10 pg methylated ismark/) . The twoWe recently demonstrated that many features of the CGI system are shared across vertebrate species .This ledR2 > 0.95, Supplementary Figure S1), further supporting the robustness of our approach  coupled to massively parallel sequencing to isolate regions of the genome containing hypomethylated DNA, and mapped these regions onto the mouse genome plus human chromosome 21 ,38. We capproach . To ensuTo investigate how HMRs form on human chromosome 21 in the mouse nuclear environment, we identified HMRs in developmentally distinct testis and liver tissue from the Tc1 mouse and directly compared these to HMRs formed on chromosome 21 in the corresponding human tissues . This reAlthough HMRs on chromosome 21 were for the most part appropriately specified in the Tc1 mouse, we observed a number of locations where DNA methylation was gained or lost in the Tc1 mouse when compared to the same locus in human   Figure –E. At th) Figure –E. Inter) Figure , upper, ) Figure , lower sThe identification of ssHMRs on human chromosome 21 in the human and mouse nuclear environments provided a unique opportunity to examine these differences in detail and discover the mechanisms that specify these methylation patterns. Given the historical relationship between CGIs and gene promoters, we first examined whether ssHMRs were associated with gene transcriptional start sites (TSSs). This revealed that ssHMRs are infrequently associated with TSSs (38/244 for liver and 30/209 for testis), and that the majority of these sites are located away from gene promoters Figure . FurtherGiven the overrepresentation of ssHMRs in regions of the genome distal to TSSs, we examined in more detail the DNA sequence features at these sites to understand if this could explain their species-specific methylation patterns and provide insight into the mechanisms shaping their methylation state. We initially focused on CpG dinucleotide frequency and GC content, based on our previous observations that these nucleotide features are almost universally elevated amongst regions in vertebrate genomes that lack CpG methylation . InitialCpG and GC-rich sequences that are methylated in human tissues are often associated with repetitive or parasitic DNA elements . TargeteElevated nucleotide features at young human repetitive sequences appear to contribute to their hypomethylated state in the Tc1 mouse, however this cannot explain how a subset of human HMRs on human chromosome 21 acquire DNA methylation in the mouse nuclear environment. To understand the features that drive formation of these human-specific HMRs we again examined their underlying nucleotide features. In contrast to Tc1 mouse-specific HMRs, human-specific HMRs were characterized by CpG density and GC content above the genome average, but significantly below those found at classical CGI elements Figure –B. This WDR4 gene is characterized by HNF4A and CEBPA binding events that only occur in the human liver, and this corresponds to a human-specific HMR  of these sites overlapped with a Tc1-unique transcription factor binding event Figure . For exad Figure  and F. Tprofiles ,22,23.Although it is clear in the Tc1 mouse that a subset of sites on human chromosome 21 can exist in alternative methylation states depending on the host species, the majority of HMRs throughout the human chromosome form completely normally Figure –E. This To test this hypothesis, we introduced large chromosomal fragments of mouse DNA into the zebrafish genome and used BioCAP-seq to examine how the resulting methylation profiles compared to the profiles observed in mouse tissues. To achieve this, four mouse bacterial artificial chromosomes (mBACs) each containing approximately 200 kb of genomic sequence were introduced into the genome of the fertilized zebrafish zygote by Tol2-mediated transposition . When weInterestingly, a small number of new HMRs were formed on the mouse BACs integrated into zebrafish, much like the species-specific HMRs that were observed in the Tc1 mouse. When we examined these sites in more detail they again corresponded to CpG and GC rich regions within the mBAC including an exonic region of the Zfp623 gene Figure . Importain vivo across distinct vertebrate organisms that have undergone extensive divergent evolution, we find that nucleotide content, in particular CpG and GC richness, is a central feature shaping HMR formation. Furthermore, the fact that most mouse HMRs, when situated in their normal genomic context, formed accurately in zebrafish  clusters that encode a historical memory of transposition events from invading retroviruses and recruit PIWI proteins to endogenous transposons resulting in their methylation ,57. Altein vivo  with the accession number GSE72208.


P < 0.001), receiving all vaccinations at age-appropriate times , and less than 7 months undervaccinated . Combination vaccines were associated with improved completion and compliance and should be encouraged among children who are undervaccinated or who received single-antigen vaccines only.Vaccination at age-appropriate intervals increases protection against morbidity and mortality; however, compliance rates among children remain low partly due to a complicated vaccination schedule. Use of combination vaccines reduces the number of injections per visit; however, there is limited evidence quantifying the effect of combination vaccines on vaccination rates. To examine how combination vaccines impact childhood completion (receipt of recommended doses) and compliance (receipt of age-appropriate vaccinations) rates, this study analyzed vaccination data from the 2012 National Immunization Survey (NIS), a nationally representative cross-sectional survey of caregivers of children aged 24 to 35 months in the United States. Vaccines were categorized as combination or single antigen. Vaccine completion was measured at ages 8, 18, and 24 months. Vaccine compliance and time undervaccinated were measured at 24 months. Children who received at least 1 combination vaccine (86%) had a higher completion rate (69%) and compliance with the full vaccine series (4:3:1:3:3:1:4 series) at 24 months (24%) than those who received only single-antigen vaccines . Receipt of combination vaccine was associated with an increased likelihood of completing all recommended vaccinations at 24 months (odds ratio [OR] = 2.5;  Despite the Advisory Committee on Immunization Practices' (ACIP's) recommendation that children receive numerous vaccinations by 2 y of age,Several studies have quantified the effects of hepatitis B combination vaccines on the timely receipt of childhood vaccinations in the United States (US).The objective of this study was to examine the effect of all currently available combination vaccines on completion of the recommended number of vaccine doses between birth and 2 y of age and to determine the age-appropriate receipt of each dose of childhood vaccination (compliance) among a nationally representative sample of children in the US.A description of the study sample is presented in P = 0.007), were married , graduated college , and had household incomes above the poverty line .Demographic differences were observed between children who received at least 1 combination vaccine compared with those who received single-antigen vaccines only. Among children who received single-antigen vaccines only, a greater percentage of mothers were aged over 30 y  higher completion rates compared with those who received single-antigen vaccines only  higher from those who received single-antigen vaccines only (P < 0.001). The largest statistically significant differences were observed for IPV (22%), HepB (22%), and varicella (19%). Children receiving combination vaccines had a higher compliance rate for the 4:3:1:3:3:1:4 series compared with those receiving single-antigen vaccines only . For most vaccines, the mean number of days undervaccinated among children who received combination vaccines was significantly lower than among those who received single-antigen vaccines only.Compliance rates for children who received combination vaccines was significantly . Similarly, compared with children receiving single-antigen vaccines only those receiving at least 1 combination vaccine were 2.2 times more likely to receive all vaccines on time and 2.4 times more likely to have less time undervaccinated (less than 7 months).Findings from multivariable logistic regression on 3 completion and compliance outcomes  indicated receiving combination vaccines was significantly associated with improved outcomes after controlling for individual, household, and provider characteristics . For theThe present study sought to examine the effect of combination vaccines on completion and compliance of childhood vaccination in the US using a nationally representative survey of households with children. Among children who received at least 1 classifiable vaccine by 24 months of age, we found 86% received at least 1 combination vaccine. The remaining 14% of the sample received single-antigen vaccines with no evidence of a combination vaccine. Children who received combination vaccines were demographically different than those who received single-antigen vaccine only. Specifically, children with single-antigen only vaccines had a significantly higher proportion of mothers who were married, graduated college, and were living above the poverty level. Prior studies have found that parents who refuse or intentionally delay vaccines tend to belong to households with higher income, have married mothers with college educations, and are covered by private health insurance.P < 0.05). Although the 2012 NIS does not include variables allowing direct measurement of vaccine refusal or delay, we anticipate a proportion of children who were undervaccinated or delayed vaccination could be attributed to vaccine hesitancy.Factors contributing to parental vaccine hesitancy  have been widely examined. Evidence suggests vaccine refusal and delay may be attributed to perceived risks, perceived benefits, and vaccine safety or efficacy concerns.The use of combination vaccines is suggested as an effective strategy for increasing vaccination rates.Furthermore, findings from the current study revealed that children receiving at least 1 combination vaccine had better compliance rates compared with those who received single-antigen vaccines only. Low compliance among children who received single-antigen vaccines only may be in part attributed to fears of simultaneous vaccination. Prior evidence suggests that fears concerning simultaneous vaccination may lead to vaccine avoidance or delay.Adjusted logistic regression models demonstrated more than a 2-fold increase in the likelihood of being complete or compliant with the vaccine series by 24 months for children who received combination vaccines compared with those who received single- antigen vaccines only. These findings are consistent with those demonstrated in prior studies that compared compliance between children who received specific combination vaccines and single-antigen vaccines. Happe and colleagues (HAPPE 2007)Our study had several limitations, primarily related to restrictions of the NIS data set. Due to the use of telephone survey methods to identify household respondents and vaccination providers to collect information on vaccination history, estimates obtained from the NIS are susceptible to under-, inaccurate, or incomplete reporting. As completion and compliance measures rely on household- or provider-reported data, there is a possibility of bias in our measurements. Furthermore, the NIS lacks assessment of knowledge, attitudes, beliefs, healthcare seeking behavior, or other factors  related to vaccination that could provide a better understanding of reasons for administration of combination vaccines, receipt of single-antigen vaccines, and/or general vaccine hesitancy. As the 2 cohorts appear demographically different, future research should explore vaccine hesitancy as a potential confounder or moderator of combination vaccine uptake.The present study fills a critical knowledge gap by evaluating the outcomes associated with combination vaccines. Prior studies that compared combination vaccines with single-antigen vaccines did not evaluate the effect of non–hepatitis B combination vaccines on compliance measures. Moreover, prior studies had not evaluated the effect of combination vaccines on completion and compliance of the full vaccination series recommended by the ACIP. Therefore, findings from the current study provide a more comprehensive understanding of the association between receipt of combination vaccines and completion or compliance outcomes. As vaccine compliance rates among children remain low in US, findings from this study suggest that combination vaccines should be encouraged for some children who are undervaccinated or are receiving single-antigen vaccines to increase compliance. Among the proportion of children with vaccine hesitant parents, particularly those with vaccine safety concerns, new strategies toward increasing vaccination are needed. Findings from the present study indicate differences in compliance between the combination and single-antigen cohorts for vaccines not included in a combination vaccine , suggesting issues other than simultaneous vaccination may impact vaccine delay/refusal. Interventions aimed at reducing vaccine hesitancy have been widely published; however, a recent systematic literature review found many interventions were not designed or powered to assess impact on vaccine refusal or delay.We used data from the 2012 National Immunization Survey (NIS) Public Use File.From an unweighted sample of 25 736 children, we selected data for children who had complete survey responses from their caregivers, resided in the US (excluding the US Virgin Islands), and had adequate vaccination data supplied by their vaccine providers. Adequate vaccination data are a variable existing in the NIS denoting whether a child had missing or incomplete provider-reported vaccination data due to a lack of parental consent to contact the child's immunization provider(s), provider nonresponse, or incomplete provider-reported data. Excluding children with inadequate provider-reported vaccinations is a method used by the CDC when calculating annual vaccination completion using NIS data.Based on ACIP's 2012 recommendations,Haemophilus influenzae type b vaccine (Hib) doses was assessed based on the product administered. For example, a child's vaccination for rotavirus was considered complete if 2 doses of Rotarix (GSK Vaccines) or 3 doses of RotaTeq  were administered. These algorithms are detailed in the published vaccine schedule.Completion of rotavirus vaccine and Although influenza and hepatitis A vaccines are also recommended by the ACIP, we did not assess them in the present study. The NIS data do not provide vaccination dates, thus we were not able to determine whether the seasonal influenza vaccine was administered during the influenza season, which is a criterion for administration. The second dose of hepatitis A vaccine should be administered between 6 and 18 months following the first dose at age 12 months or later; therefore, the second dose may be administered after 24 months—falling outside of our study period. As we could not capture appropriate completion and compliance of 2 doses of hepatitis A vaccine, it was not included in the present analysis.Vaccine compliance is a measure of the timeliness of vaccine administration. The ACIP suggests specific ages and intervals at which vaccines should be administered to maximize effectiveness.Additionally, we measured compliance by calculating the number of days that a child was undervaccinated. This method has been proposed in several prior vaccine compliance studies.The ACIP allows early vaccination as “age-appropriate” if received within a 4-day grace period before the minimum age for the dose.We categorized all vaccines into 2 mutually exclusive groups: combination or single antigen. Although vaccines such as DTaP include multiple components in a single syringe, they were grouped with single-antigen vaccines. Preliminary review of the data indicated that no participants received the individual components of DTaP as single-antigen vaccines, and a small number of participants received a single-antigen measles vaccine; therefore, we anticipated that patterns of administration of DTaP and measles, mumps, and rubella (MMR) vaccines may be closer to those of true single-antigen vaccines  than to those of combination vaccines .After each vaccine was coded as combination or single antigen, the vaccination history of each child was reviewed to determine whether each child received at least 1 combination vaccine or single-antigen vaccines only. Next, a binary categorical measure of vaccine type was created to flag each child into 2 mutually exclusive groups: received at least 1 combination vaccine or received no combination vaccines . This categorical measure of vaccination type was used to determine differences in vaccination completion and compliance between children who received at least 1 combination vaccine and those who received single-antigen vaccines only.Bivariate analysis included evaluation of completion and compliance rates for the overall population and stratified by vaccination type. The Rao-Scott F-adjusted chi-square statistic was used to determine the differences between completion and compliance rates by vaccination type. This statistic is adjusted for complex survey data and provides a more conservative interpretation than the Wald chi-square statistic.Multivariable analysis included logistic regressions on 3 vaccination outcomes: completed vaccines series by 24 months (completion), received all vaccines on time (compliance), and undervaccinated for less than 7 months (compliance). These logistic regression models evaluated the effect of receipt of at least 1 combination vaccine on vaccine completion or compliance after adjusting for demographic characteristics such as maternal age, maternal marital status, maternal education, poverty status, enrollment in Medicaid or the State Children's Health Insurance Program (SCHIP), and provider variables including number of vaccination providers and type of vaccination providers .P values were calculated using SAS statistical software, version 9.3 . A P value less than 0.05 was used as an indicator that a difference between groups may exist. SAS survey procedures and domain analysis techniques were used to calculate weighted values and correct standard errors per NIS data user guidance.Estimated frequencies, percentages, means, standard errors, medians, odds ratios, confidence intervals, and


Many glioblastoma patients suffer from seizures why they are treated with antiepileptic agents. Valproic acid (VPA) is a histone deacetylase inhibitor that apart from its anticonvulsive effects in some retrospective studies has been suggested to lead to a superior outcome of glioblastoma patients. However, the exact molecular effects of VPA treatment on glioblastoma cells have not yet been deciphered. We treated glioblastoma cells with VPA, recorded the functional effects of this treatment and performed a global and unbiased next generation sequencing study on the chromatin (ChIP) and RNA level. 1) VPA treatment clearly sensitized glioma cells to temozolomide: A protruding VPA-induced molecular feature in this context was the transcriptional upregulation/reexpression of numerous solute carrier (SLC) transporters that was also reflected by euchromatinization on the histone level and a reexpression of SLC transporters in human biopsy samples after VPA treatment. DNA repair genes were adversely reduced. 2) VPA treatment, however, also reduced cell proliferation in temozolomide-naive cells: On the molecular level in this context we observed a transcriptional upregulation/reexpression and euchromatinization of several glioblastoma relevant tumor suppressor genes and a reduction of stemness markers, while transcriptional subtype classification  remained unaltered. Taken together, these findings argue for both temozolomide-dependent and -independent effects of VPA. VPA might increase the uptake of temozolomide and simultaneously lead to a less malignant glioblastoma phenotype. From a mere molecular perspective these findings might indicate a surplus value of VPA in glioblastoma therapy and could therefore contribute an additional ratio for clinical decision making. Glioblastoma multiforme is the most common malignant brain tumor. The standard therapy consists of surgical removal and concomitant radiochemotherapy followed by adjuvant chemotherapy with temozolomide . But desValproic acid (VPA), for example, is an antiepileptic drug that is often used to treat seizures in glioblastoma patients. Interestingly, a number of retrospective analyses supported a disease course-modifying role of valproic acid in glioma patients –9. As suin vitro. It could be demonstrated that VPA inhibits cell proliferation by causing cell-cycle arrest in the G1 and/or G2 phase and that it induces differentiation and/or apoptosis in cancer cells [MGMT (O-6-methylguanine-DNA methyltransferase) and to sensitize human glioma cells to temozolomide and irradiation [Several cell biological studies have investigated VPA effects on tumor cells er cells , 11. Valer cells  and decrer cells . Furtheradiation , 15.Nevertheless, there is yet little systematic understanding of the exact VPA mode of action and -in particular- the molecular correlates associated with it. We thus treated a large cohort of classical adherent glioblastoma and primary glioblastoma stem cell lines with VPA, recorded the functional implications of this treatment and performed global and unbiased next generation sequencing (NGS) analysis on the RNA level. As VPA functions as a histone-deacetylase inhibitor (HDACi) and specifically inhibits HDAC classes I and IIa  we also Despite a recent metaanalysis of prospective clinical trials that could not confirm a beneficial effect of VPA on outcome in newly diagnosed glioblastoma patients , there i50 values.First, we aimed to investigate the functional implications of VPA treatment on glioma cells. We therefore treated the six established glioblastoma cell lines and HS683 with VPA and subsequently performed temozolomide chemosensitivity assays. All seven cell lines were sensitized to temozolomide Figure . Five ceWe then subjected the seven glioblastoma stem cell lines to identical experimental conditions. However, three of the lines, i.e. NCH1425, NCH601 and NCH636, were not suited for functional assays due to the fact that they were very sensitive to dissociation procedures necessary for cell counting and seeding. Hence, they were omitted from the experiment. In the four remaining stem cell lines that could be tested, we observed a similar sensitization to temozolomide after VPA treatment as for the established glioma cell lines Figure .We next wanted to know whether VPA treatment would have effects on tumor cell proliferation also irrespectively of temozolomide in a chemotherapy-naive situation. Indeed, in the broad majority of the investigated cell lines  VPA treatment significantly reduced proliferation  based on the RPKM (reads per kilobase per million mapped reads) values of the sequenced samples Figure . SamplesThis 497-gene expression signature was then subjected to GO term analyses. GO terms containing the designation “transport” were most prominently enriched  that were upregulated on the mRNA level with the highest fold change (≥5) and had concomitant promoter euchromatinization on ChIP-Seq. All four transporters after VPA treatment were overexpressed in the majority of cell lines, partly up to more than 100-fold .For two of the transporters (SLC17A7 and SCL25A27) antibodies were available that worked in formalin-fixed paraffin-embedded tissue. We stained matched pairs of patient biopsy samples (n=6) that allowed for the comparison of SLC transporter expression prior and after VPA treatment. Indeed, for SLC17A7 we detected a reexpression in three out of six patients  for the siCTRL transfection and set this to 1. We then assessed the VPA-induced effects in response to temozolomide with the respective targeting siRNAs and normalized them to siCTRL. If for a defined siRNA the VPA-induced temozolomide sensitization was stronger compared to siCTRL this would lead to a relative proliferation value of <1 indicating an additional sensitization induced by the siRNA knockdown. If the VPA-induced temozolomide sensitization in the context of a defined siRNA was weaker compared to siRNA control this would lead to a relative proliferation value >1 indicating a desensitization by the respective siRNA knockdown and thus the presumed rescue effect  close to the expected ICt Figure . Here, fSince this rather complex experimental setting did not allow for detecting a clear rescue effect we next wanted to know whether the SLC transporter knockdown in a situation uninfluenced by VPA would lead to temozolomide desensitization. This would be indicative for the transporters being involved in the uptake of temozolomide. Here, results were still rather inconsistent but knockdown for a higher number of transporters led to a slightly enhanced relative proliferation  after VPA treatment. H3ac binding to the MGMT promoter was significantly increased (p<0.01) in six cell lines but this cannot explain for temozolomide sensitization. Taken together, these findings do not argue for a major relevance of MGMT in the VPA response.As MGMT had been linked to the VPA response , we assees Table . Except PARP2 and PARP3 were most frequently downregulated by VPA treatment since they showed a significant downregulation (p<0.01) in ten and eleven out of the 14 cell lines, respectively. Amongst the double-strand break repair genes, NBN and RAD51 exhibited a significant downregulation in the majority of cell lines . The expression of mismatch repair genes in comparison (except for MLH1) was downregulated in much lower frequencies in only individual cell lines.Apart from upregulation of SLC transporters, GSEA also pointed towards a downregulation of DNA repair genes after VPA treatment Figure . EuchromCDKN1A (p21) and SFRP1 appeared to be majorly affected, showing significant upregulation (p<0.01) in eight cell lines, respectively. Reexpression of CDKN1A was validated by q-RT-PCR analyses  . AccordiVPA, an antiepileptic drug used to treat seizures in brain-tumor patients, has been suggested to have anti-cancer effects but its exact molecular modes of action have not yet been deciphered. VPA is also a histone-deacetylase inhibitor . We therin vitro studies [First, we assessed the functional implications of VPA treatment on our cell lines. Indeed, in all lines studied VPA treatment sensitized glioma cells to temozolomide. This finding for adherent glioma cell lines had been reported in a similar fashion in a number of preceding  studies , 15, butComparing the molecular profiles before and after VPA treatment we found that GO terms with the functional annotation “transport” were associated with the VPA response signature. Indeed, numerous SLC transporters were transcriptionally upregulated and euchromatinized following VPA treatment. The solute carrier (SLC) group of membrane transporters comprises a family of more than 300 membrane-bound transporters . SLC traGiven this apparent association between VPA-induced chemosensitization and SLC transporter upregulation we followed up functionally on these relationships. We first aimed to rescue the VPA-induced chemosensitization by siRNA-mediated transporter knockdown. Though leading to a moderate chemodesensitization for individual transporters these effects did not appear convincingly strong and consistent Figure . HoweverMGMT [MGMT expression and promoter methylation were hardly altered by VPA treatment and the MGMT euchromatinization we observed could not explain for temozolomide sensitization. Taken together, our analyses do not support a major relevance of MGMT as mediator of the observed VPA response. In glioblastoma patients, however, also DNA repair mechanisms other than MGMT may modify the temozolomide response [APEX1 has been reported to enhance the cytotoxic effects of temozolomide [NBN resulted in a sensitization to temozolomide in melanoma cells [RAD51 induced a temozolomide sensitizing effect in glioma cells [MSH2 and MSH6, has been described to evolve under therapy with alkylating agents and would rather mediate opposite effects, i.e. temozolomide resistance [MLH1), however, were only reduced in individual cell lines.As others had shown that VPA downregulated the expression of MGMT  and thisresponse . Of notezolomide , 27. Alszolomide , 29. Inama cells  and the ma cells . We founsistance , 33. TheCDKN1A (p21) and SFRP1 being the most frequently upregulated genes on the mRNA level. The finding of CDKN1A reexpression is in line with a preceding publication that describes upregulation of this gene after VPA treatment [SFRP1 encodes a WNT inhibitor the expression of which has been shown to be of positive prognostic relevance in gliomas [Our functional analyses revealed that VPA treatment had also direct (temozolomide-independent) effects on tumor cell proliferation. A molecular correlate that might explain for this finding was the euchromatinization and upregulation of various well-established tumor suppressor genes, with reatment . SFRP1 e gliomas . Both ge gliomas .In summary, we here present a comprehensive molecular next generation sequencing study deciphering the global molecular VPA effects on the epigenetic (histone) and transcriptional level. We find that the VPA effects are manifold including molecular alterations that might directly enhance the temozolomide response and others that might induce temozolomide-independent favorable effects on glioma biology. Thus, our study from a molecular point of view highlights potential anti-tumorigenic modes of action of VPA in glioma cells. The question remains whether this also translates into relevant clinical effects as a very recent pooled analysis of prospective clinical trials (retrospective in nature in respect to VPA though) could not retrace an association between VPA use and improved survival outcomes in patients with newly diagnosed glioblastoma . FactorsIn case a prospective randomized controlled clinical trial on the use of VPA in glioblastomas should still be intended, our analyses reveal that there a) will be no simple molecular read-out that could be used for stratification or an upfront identification of patients most likely to benefit from standard therapy plus VPA and that b) also the underlying molecular set-up of the individual patient may well influence the response towards the agent. For the study design it thus would be the challenge to best possibly control for such underlying molecular factors and to keep patient groups as homogeneous as possible.2, DMEM, 10% FCS, 1% penicillin/streptomycin). In addition, seven human primary stem cell-like glioblastoma cell lines  [2, DMEM/Ham's F-12 medium, 1% penicillin/streptomycin, 1% L-glutamine. Directly before use, stem cell medium was prepared freshly by adding 20% BIT admixture supplement , 0.02% epidermal growth factor (EGF)  and 0.02% basic fibroblast growth factor 2 (bFGF2) (ReliaTech). Valproic acid sodium salt  was dissolved in distilled water and subsequently diluted freshly in complete DMEM. Cells were treated with 7.5 mM VPA [Six established adherent glioblastoma cell lines  and HS683 (derived from an anaplastic oligodendroglioma) were cultured under standard conditions . Expression of candidate gene transcripts was validated by real-time quantitative RT-PCR based on the SensiFASTTM SYBR Hi-Rox Kit  with the StepOnePlusTM sequence detection system (Life Technologies). Fold expression changes relative to non-neoplastic brain tissue were calculated with the ΔΔCT method [GAPDH  as the reference transcript  was used according to the manufacturer's protocol to extract RNA from untreated and VPA-treated cells, respectively. Subsequent cDNA synthesis from one microgram total RNA was performed using random hexamer primers  and the SuperScriptT method  using GA6 cells  were incubated with 1% formaldehyde  for 10 minutes to cross-link the DNA with proteins. Afterwards, cells were resuspended in freshly prepared swelling buffer. Isolated nuclei were then further processed using a commercial ChIP assay kit  following the manufacturer's recommendations. After resuspension in SDS lysis buffer, lysates were sonicated with an ultra-sonicator  to shear the DNA into 200-800 bp fragments. Sonication was followed by centrifugation and samples were diluted in ChIP dilution buffer and precleared with salmon sperm DNA/protein A agarose beads. Immunoprecipitation with antibodies against acetylated histone H3 (H3ac) was performed overnight at 4°C, rabbit anti-human IgG fraction served as a negative control. Then, the antibody-histone-DNA complexes were collected and histone-DNA complexes were eluted in freshly prepared elution buffer. After reversion of the cross-link, the DNA was recovered by phenol/chloroform extraction and ethanol precipitation.For chromatin immunoprecipitation 10H3ac-immunoprecipitated DNA from untreated and treated cells was used to generate libraries for NGS using the TruSeq® ChIP Sample Preparation Kit  according to the manufacturer's instructions. Isolated RNA from untreated and VPA-treated cells was converted into libraries of template molecules suitable for NGS using the TruSeq® RNA Sample Preparation Kit v2 (Illumina). All libraries were quantified using the KAPA SYBR FAST ABI Prism Library Quantification Kit . Equimolar amounts of each library were used for cluster generation on the cBot with the TruSeq SR Cluster Kit v3 (Illumina). The sequencing run was performed on a HiSeq 1000 instrument (Illumina) using the indexed, 50 cycles single read (SR) protocol and the TruSeq SBS v3 Kit (Illumina). Image analysis and base calling resulted in .bcl files which were then converted into .fastq files by the CASAVA1.8.2 software. Analysis of NGS data was performed using the Genomatix software . First, the .fastq files were mapped to the human genome hg19 (annotation based on ElDorado 12-2012) using the Genomatix Mining Station. Then, all unique hits were further processed using the Genomatix Genome Analyzer  was performed in order to determine whether a priori defined sets of genes were over- or underrepresented in our gene expression signatures. Glioblastoma signature genes were taken from [Principal component analysis (PCA) was based on the top 1,800 most variable genes that were calculated from the RPKM (reads per kilobase per million mapped reads) expression values. A biplot was then created with custom R scripts and the package ggbiplot. GO term analysis was performed using the DAVID  bioinformatics resources as described elsewhere  and a coken from .50) value was determined as the concentration resulting in a 50% growth reduction compared to control cell growth (i.e. cells that did not obtain temozolomide). Statistical analyses were performed using the GraphPad Prism software (version 6). Fluorescence intensities were log-transformed, normalized and then fitted with the methods of least squares and variable slope.Cells were seeded in triplicates in 96-well plates at a density of 500 cells in 100 μl per well. 24 h later, the medium was replaced by fresh medium with or without 7.5 mM valproic acid, respectively. After another 24 h, various concentrations of temozolomide  were added. Cell viability was measured 72 h later using resazurin  as described elsewhere –45. The MGMT promoter DNA from untreated and VPA-treated samples was assessed via MethyQESD (methylation-quantification of endonuclease-resistant DNA) as described before [Methylation of d before .TM transfection reagent  according to the manufacturer's protocol. Cells were seeded in triplicates in 96-well plates at a density of 3,000 cells in 100 μl per well. 24 h later, half of the cells were treated with 7.5 mM VPA for 24 h. Then, 25 nM of siRNAs were transfected in medium with or without valproic acid, respectively. The next day, temozolomide at a concentration of 1,500 μM was added and cell viability was measured 72 h later via the resazurin assay.Transfections with small interfering RNAs were performed using the DharmaFECTTM+ Dual Link System-HRP  was used for detection of antibody binding according to the manufacturer's protocol. Afterwards, nuclei in the immunostained sections were counterstained with haematoxylin.Matched pairs of formalin-fixed and paraffin-embedded tissue samples from brain tumor patients before and after VPA treatment were selected from the tumor tissue archive of the Regensburg Department of Neuropathology and investigated according to protocols approved by the institutional review board. Immunohistochemical staining was performed following a standard protocol . Briefly