CREB-H (64-fold) as well as ER membrane components INSIG and gp78, which modulate cholesterol synthesis while downregulating over 20 components mostly associated with ERAD and FOLD. We hypothesize that this expression pattern is associated with an expanding ER with downregulation of active degradation by ERAD and apoptosis as the cell attempts to handle abundant viral glycoprotein synthesis.Varicella-zoster virus (VZV) is a human herpesvirus that spreads to children as varicella or chicken pox. The virus then establishes latency in the nervous system and re-emerges, typically decades later, as zoster or shingles. We have reported previously that VZV induces autophagy in infected cells as well as exhibiting evidence of the Unfolded Protein Response (UPR): XBP1 splicing, a greatly expanded Endoplasmic Reticulum (ER) and CHOP expression. Herein we report the results of a UPR specific PCR array that measures the levels of mRNA of 84 different components of the UPR in VZV infected cells as compared to tunicamycin treated cells as a positive control and uninfected, untreated cells as a negative control. Tunicamycin is a mixture of chemicals that inhibits N-linked glycosylation in the ER with resultant protein misfolding and the UPR. We found that VZV differentially induces the UPR when compared to tunicamycin treatment. For example, tunicamycin treatment moderately increased (8-fold) roughly half of the array elements while downregulating only three (one ERAD and two FOLD components). VZV infection on the other hand upregulated 33 components including a little described stress sensor  VZV is a human pathogen that spreads to children as varicella or chicken pox and re-emerges later as zoster or shingles Ross, . VZV is Varicella infection, within its natural human host, spreads from the nasopharynx via infection of a limited number of T cells that home to the skin epidermis : XBP1 splicing and a greatly expanded ER  or VZV ORF62 (pCMV_IE62) under the CMV promoter as described previously  to form ΔCT values which were then used to calculate averages and standard deviations between triplicate measurements. Subsequent ΔΔCT values were calculated by differences between averages of VZV infected ΔCT values or tunicamycin treated ΔCT values with the average uninfected ΔCT values. Uncertainties correspond to propagation of errors using standard deviations between the uninfected and infected or tunicamycin averages.Total RNA was extracted from uninfected, tunicamycin treated and VZV infected fibroblast cells in six well plates at the given time points using the RNEasy mini kit (Qiagen). RNA quality and quantity was assayed by UV spectroscopy using a NanoDrop spectrometer. Both A260/A280 and A260/A230 ratios were within 20% of 2.0 and infected cells from a six well plate well (6.5 sq cm) yielded approximately 3 μg of RNA in 60 μl. Further, the RNA was electrophoresed in an Agilent Bioanalyzer 2100 (Agilent) and yielded RIN values within 20% of 10. Polyadenylated RNA was converted to cDNA using anchored Oligo(dT) primers and the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen) to yield approximately 20 ng of cDNA. The entire cDNA sample from one well of cells was mixed into 1 ml of 1× diluted Power SYBR Green Master Mix (ABI) and split into all wells of a SA Biosciences UPR PCR array (Life Technologies) with a multichannel pipettor (25 μl per well). The measurements were carried out in triplicate using cDNA from three of the original six wells in the plate for all types of samples using a Model 7000 real time PCR instrument (ABI). The resulting PCR results were processed using the SDS 1.2.3 software (Applied Biosystems). CTo confirm measurements from the UPR specific PCR array, several RT-PCR measurements were carried out using the following primers: BiP: forward 5′-CCC CAA CTG GTG AAG AGG AT-3′ and reverse 5′-GCA GTA AAC AGC CGC TTA GG-3′; DNAJB9/ERDj4: forward 5′-ACA TCT GTG ACT TGC GTT GC-3′ and reverse 5′-TGG GCA ATA AAA CCA TTT CC-3′; CREBH: forward 5′-GGG AGA CGA GCT GTG AGC-3′ and reverse 5′-TGT CTG AGT GTC GGT TCC TG-3′; PERK: forward 5′-GCC TAA GGA GGT AGC AGC AA-3′ and reverse 5′-GGG ACA AAA ATG GAG TCA GC-3′.Murine MAb antibodies to VZV gE (3B3) and IE62 (5C6) produced in our laboratory were used in addition to a rabbit polyclonal antibody to LC3B (Santa Cruz Biotech sc-28266).Samples of infected and uninfected cells were prepared for confocal microscopy by methods described previously  have been described in earlier papers in which we were investigating VZV glycoprotein biosynthesis  was obtained in powder form from Molecular Probes (D-273) and dissolved (0.7 mg/ml) in ethanol  was added to warm cell culture medium; this medium was applied to live cells for 30 min, then rinsed 2× with PBS and processed for fluorescent microscopy as described above.DiOCWithin cell culture, VZV is entirely cell associated with no release of cell-free virus . This 96 well plate consists of 84 wells containing primers to the 3′ Untranslated Region (UTR) of transcripts associated with the UPR and the remaining 12 wells containing primers to housekeeping genes and PCR and cDNA quality control wells. Table T values were normalized so that in each case the housekeeping gene transcripts measured CT average was 16 and then the triplicate measurements were averaged and standard deviations computed to generate ΔCT. Differences between the uninfected ΔCT and those associated with TM treated and VZV infected cell transcript measurements were then calculated to form the final measurements ΔΔCT listed in Table CHOP (pink). By contrast, only 43 of the UPR genes are upregulated in VZV infected cells. In particular, those genes most upregulated such as CREB3L3/CREBH (light blue) are more upregulated than after TM treatment. VZV infected cells also upregulated the LIPID transcripts AMFR/gp78 and INSIG (green) while downregulating a number of ERAD components such as UBXN4/erasin and EDEM3 (red). These differences will be considered by group in the subsequent sections.Gene transcripts were measured in uninfected human fibroblasts, tunicamycin (TM) treated fibroblasts and VZV infected fibroblasts. Each measurement was done in triplicate. The measured CPERK, IRE1α and ATF6 but also two CREB proteins (CREB3/LUMAN and CREBH) as well as primers to the Golgi resident proteases S1P and S2P that activate AT6 and the CREB proteins by cleavage  than in TM treated cells . However, the downregulation of PERK in VZV infected cells was not confirmed.In order to confirm the results of the UPR specific PCR array, we carried out qPCR measurements using primers to K Figure  in TM trHSPA5/BiP and SIL1/BaP; five DNAJ HSP-40 homologs including DNAJB9/ERdj4 and DNAJC10/ERdj5; twelve wells contain primers to transcripts encoding ER lumen folding components including CALR and CANX; three components of the folding chaperonin complex and finally four components in the ER membrane including RPN1 and SEC63. Many of these transcripts encode proteins which assist secretory protein folding but also sense misfolded proteins in the ER . However, we also reassessed the regulation of the ER-co-chaperone DNAJC10/ERdj5 and found a correlation with the UPR-specific array (data not shown).VZV infected cells exhibited very uneven transcription of folding chaperones (FOLD) while TM treatment robustly upregulated transcription of these chaperones particularly P Figure . Measures Figure  and founOS9, PPIA and SELS along with a number of FOLD transcripts), trimming of mannose residues prior to recognition by E3 ubiquitin ligases (EDEM1 and EDEM3), recognition of misfolded proteins by E3 ubiquition ligases , exportation to the cytosolic side of the ER membrane (SEC62) where the VCP/p97 complex poly-ubiquitinates protein substrates before extracting/clipping the protein from the membrane to be ultimately degraded in the cytosol by the proteasome  in VZV infected cells as compared to the value in TM treated cells . We therefore conclude that expression of a single VZV glycoprotein gene in cells is sufficient to activate the CREBH arm of the UPR.In 2011, we found that transfecting cells with VZV glycoprotein genes led to increased autophagosome production and inflation of the ER. Transfection with VZV IE62 led to neither increased autophagosomes nor a larger ER. Therefore, we measured by qPCR whether CREBH and BiP transcription was increased by transfection of a glycoprotein vs. a non-glycoprotein that is also the major transactivator encoded by VZV. Transfection with a plasmid encoding VZV gE under the CMV immediate early promoter led to approximately 10% of transfected cells Figure  while tr Figures  whereas CREBH. At the same time, VZV infected cells showed decreased transcription of genes associated with ERAD and apoptosis. We hypothesize that this transcriptional profile is compatible with the infected cell attempting to accommodate the influx of viral glycoproteins by greatly increasing the capacity of the ER. For example, increased transcription of AMFR (gp78) and INSIG is associated with a decrease in cholesterol synthesis via degradation of HMG-CoA reductase, an enzyme necessary for cholesterol synthesis  The choice of primers in the PCR array vs. those used in the qPCR measurements or (ii) the asynchronous nature of VZV infection. In general, there was better agreement between tunicamycin treated values as measured by the UPR-specific PCR array and individual qPCR assays. The biggest differences were observed in values measured from VZV infected cells. The latter scenario suggests that asynchronous VZV infection may play a role; for example, the input virus is always extremely low, such that some cells within a monolayer will remain uninfected even at 72 hpi. This same scenario may explain why we have observed similar unexpected differences in experiments to measure protein expression, for example, BiP. Because of increased VZV-induced autophagy, we predicted increased BiP production in infected cells. However, we have observed variable changes in BiP protein expression following VZV infection. Nevertheless, the main conclusions of this report were confirmed by both assays, namely, the significant upregulation of CREBH as well as the more modest upregulation of the cholesterol regulator INSIG.A key question is whether the infected cell is responding to abundant viral glycoprotein expression through normal mechanisms or alternatively, does the virus encode its own proteins which manipulate the UPR. Recently, Burnett et al found that HSV ICP0 transactivated elements of the UPR and in turn was transactivated by the UPR itself via an ERSE promoter element in the HSV ICP0 gene (Burnett et al., CREBH and BiP transcription. We observed abundant VZV gE protein in the ER/Golgi after transfection. In contrast, transfection with VZV IE62, a non-glycosylated viral protein, did not lead to increased transcription of either transcript. Obviously, the IE62 protein never enters the ER/Golgi. These results confirm and expand our 2011 report that transfection with VZV glycoprotein genes resulted in increased ER size and increased autophagosome production. The UPR is known to upregulate autophagy (Yorimitsu et al., In further support of our hypothesis that abundant expression of VZV glycoproteins contributes to the activation of the UPR in a specific way that leads to an enlarged ER and increased autophagosome production, we found that transfection of the VZV gE gene led to increased CREBH and cholesterol synthesis regulation transcription and diminished ERAD transcription. The transcription patterns appeared to correlate with increasing ER capacity secondary to increasing viral glycoprotein synthesis in the infected cell. Of importance, the CREBH data were totally unexpected, based on all prior VZV research, and would never have been uncovered in the absence of the UPR array data described in this report.In summary, even though both tunicamycin treatment and VZV infection induced an UPR, the profiles of UPR related genes were different after the two analyses. The UPR in VZV infected cells exhibited greatly increased 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.


The aim of this study was to characterise the new hydrophilic fissure sealant, UltraSeal XT® hydro™ was applied to both groups of teeth which were then subjected to 2,500 thermocycles between 5 and 55°C prior to microleakage assessment by fuchsin dye penetration. The sealant was characterised by Fourier transform infra-red spectroscopy (FTIR), scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDX), and Vickers indentation test. Occlusal surfaces of extracted human molars were either conventionally acid etched (n=10), or sequentially acid etched and laser irradiated (n=10). UltraSeal XT® hydro™ is an acrylate-based sealant that achieved a degree of conversion of 50.6±2.2% and a Vickers microhardness of 24.2±1.5 under standard light curing . Fluoride ion release is negligible within a 14-day period. SEM and EDX analyses indicated that the sealant comprises irregular submicron and nano-sized silicon-, barium-, and aluminium-bearing filler phases embedded in a ductile matrix. Laser preconditioning was found to significantly reduce microleakage . The lased teeth presented enhanced surface roughness on a 50 to 100 μm scale that caused the segregation and concentration of the filler particles at the enamel-sealant interface. UltraSeal XT Laser preconditioning significantly decreased microleakage and increased enamel surface roughness, which caused zoning of the filler particles at the enamel-sealant interface. Accordingly, resin-based or glass ionomer sealants are placed over the occlusal surfaces of premolars and molars to prevent cariogenic microorganisms and fermentable organic debris from accumulating in the pits and fissures11.Approximately 90% of all dental caries arise in the occlusal pits and fissures, since these regions are anatomically defended against the remineralising flow of saliva and routine brushing13. The presence of moisture and saliva-contamination during the placement of the sealant compromise the quality of adhesion at the sealant-enamel interface, which impacts the ongoing resistance to microleakage of microorganisms. Recently introduced hydrophilic sealants, which bond effectively to moist enamel surfaces, present a distinct advantage in paediatric dentistry where patient-compliance, isolation, and moisture-control can be particularly challenging10.The majority of commercially available resin-based pit and fissure sealants are hydrophobic materials that bond to the enamel surface via micromechanically interlocking tags® hydro™ is a new moisture-tolerant, self-adhesive, light-cured, acrylate-based, hydrophilic pit, and fissure sealant which has been developed by Ultradent Products, USA4. The sealant comprises a 53 wt% mixture of inorganic filler particles that confer radiopacity. This material is reported to “chase” moisture into the pits and fissures, thus eliminating moisture-related failure associated with hydrophobic sealants4.UltraSeal XT1. Recent research has indicated that the application of laser ablation as an adjunct to phosphoric acid etching may improve the adhesion, marginal adaptation, retention, and resistance to microleakage of resin-based sealants10. This finding is not universal, and the efficacy of laser conditioning appears to depend upon the rheological and physicochemical properties of different sealants12. It is suggested that less viscous, more “flowable” materials may afford better adaptation to the rougher surfaces presented by lased enamel9.Prior to the application of a resin-based fissure sealant, the enamel surface is etched with phosphoric acid gel to enhance surface roughness and wettability and to increase porosity and surface area available for bonding® hydro™, and to investigate its resistance to microleakage in vitro. The chemical composition and degree of curing of UltraSeal XT® hydro™ were investigated by Fourier transform infra-red spectroscopy (FTIR). Scanning electron microscopy (SEM) was used to examine the fracture surface and the elemental composition was determined by energy dispersive X-ray analysis (EDX). Microhardness was evaluated using the Vickers indentation test, and fluoride-release was monitored with an ion-selective electrode.The objectives of this study were to characterise the new hydrophilic sealant, UltraSeal XTst October 2014 by the Ethical Committee of Bezmiâlem Vakif University (reference number 71306642/050-01-04/282), which was performed according to the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.Ethical approval for this project was obtained on 1® hydroTM sealant  used in this study, as listed in the safety data sheet provided by the manufacturer14, is given in ® hydro™ monomer solution that was light-cured for 20 s at 1000 mWcm-2 . The resulting cured UltraSeal XT® hydro™ discs were then characterised by FTIR, SEM, and EDX and their microhardness and fluoride-release behaviour were measured.The composition of the UltraSeal XT® hydro™ for SEM and EDX analysis were produced by crushing the cured discs between steel plates under a low-impact force. Scanning electron microscopy was carried out on the fracture surfaces of uncoated samples attached to carbon tabs on a JEOL JSM-5410 LV electron microscope with an Oxford Instruments X-MaxN EDX detector in low vacuum mode. All back-scattered and secondary electron images were obtained with an accelerating voltage of 1.0 kV at a working distance of 8.4 mm.Fracture surfaces of UltraSeal XT® hydro™ monomer solution and of the cured discs were obtained in triplicate using a Perkin Elmer Spectrum Two spectrometer with a Universal Diamond attenuated total reflectance attachment . Spectra were recorded with 16 accumulated scans between 4000 cm-1 and 450 cm-1 wavenumbers at a resolution of 4 cm-1.The FTIR spectra of the unset UltraSeal XT-1) and the invariant C=O bond (at 1717 cm-1) in the cured polymer and monomer using the following equation:The degree of conversion (DC) was estimated by comparing the ratios of the intensities of the FTIR peaks for the reactive polymerising C=C bond  and Orion 4 Star meter .Three cured UltraSeal XT® hydro™ discs using a Buehler Micromet Hardness Tester  with a load of 200 g and contact time of 2.5 s. Three measurements were taken at random points on both sides of each sample disc. Indentation diagonals were measured in micrometres with the aid of a stereomicroscope and converted to Vickers hardness numbers (VHN) using conversion tables provided by Buehler, Coventry, Warwickshire, UK.The Vickers microindentation test was carried out on 3 UltraSeal XTTwenty sound extracted human molar teeth were obtained from patients with orthodontic or periodontal problems who had tendered their informed consent. The teeth were manually debrided with scaling instruments, cleaned with pumice paste and stored in distilled water for up to four weeks. The teeth were then randomly divided into two groups. A flow diagram for the experimental procedures is given in ® hydro™, Ultradent Products, South Jordan, Utah, USA) for 20 s, rinsed and lightly air dried, as suggested by the manufacturer. The UltraSeal XT® hydro™ sealant was then applied by the same operator, according to the manufacturer’s instructions, and light cured for 20 s .The occlusal surfaces of Group I teeth (n=10) were acid etched with 35% phosphoric acid gel . The Er:YAG laser energy was applied at a density of 19 mJ cm-2, power output of 1.2 W, and pulse energy of 120 mJ using a 600 μm diameter sapphire tip with a beam spot size of 0.63 mm2 at a working distance of 8 mm at an angle of 90° under water cooling at 50 cm3min-1. The lased teeth were rinsed with water, thoroughly air dried, acid etched (as outlined above), and then lightly air dried prior to sealing with UltraSeal XT® hydro™.Group II teeth (n=10) were sequentially subjected to laser ablation and acid etching. Laser conditioning of the occlusal surfaces of Group II teeth was carried out using a 2940 nm Er:YAG laser system . Each sample was placed in 0.5% basic fuchsin dye solution for 24 h, rinsed under flowing tap water for 5 min to remove excess dye, and sectioned in the bucco-lingual direction using a water-cooled diamond saw to obtain three slices. Each of the tooth sample slices was then examined twice under a stereomicroscope  at 20x magnification by two investigators who were unaware of the pre-treatment of each sample. The microleakage scoring criteria are listed in The microleakage data were analysed using the statistical package SPSS 14.0.0 for Windows . Significant differences were evaluated using the Mann-Whitney U test (p=0.05). Inter-examiner variability was analysed with the kappa statistic, which was found to be 0.9, indicating high reproducibility.SEM and EDX analyses were carried out on the middle slices of the sectioned teeth using uncoated samples attached to carbon tabs on a JEOL JSM-5410 LV electron microscope with an Oxford Instruments X-MaxN EDX detector in low vacuum mode. All back-scattered electron images and EDX maps were obtained with an accelerating voltage of 20 kV at a working distance of 20 mm.® hydro™ are presented in Secondary electron images showing the topography of the fracture surfaces of light cured UltraSeal XT® hydro™ fracture surfaces  lists sodium monofluorophosphate (Na2PO3F) at levels below 0.2 wt%, which were found to be below the EDX detection limit for fluorine.The EDX analysis of a 100 μm by 125 μm window of a polished section of the light cured sealant indicated that this material is principally composed of carbon, oxygen, barium, silicon, aluminium, calcium, and phosphorus . Individ® hydro™ 14 lists® hydro™ solution and light cured polymerised discs are shown in 15. According to the manufacturer’s safety data sheet (14), the organic component of UltraSeal XT® hydro™ comprises a mixture of triethylene glycol dimethacrylate (TEGDMA), diurethane dimethacrylate (DUDMA), and methacrylic acid (MAA). Present in these monomers are hydrophilic carboxylic acid (-COOH), secondary amine (-NH) and carbonyl (-C=O) groups, hydrophobic alkane (-CH3) and alkene (-C=CH2) groups, and ether (-C-O-C-) groups, which are of amphiphilic character. Each of these functional groups appears in the FTIR spectra of both the unset UltraSeal XT® hydro™ solution and light cured polymerised discs , the level of sodium monofluorophosphate (Na2PO3F) in UltraSeal XT® hydro™ is lower than 0.2 wt%. This corresponds with a maximum fluoride ion concentration within the sealant of 0.0264 wt%.Light cured UltraSeal XTta sheet 14, the ® hydro™ was found to be 24.2±1.5.Microhardness provides a measure of the resistance of a sealant to plastic deformation under applied compressive forces. The Vickers hardness number (VHN) of light cured UltraSeal XTin vitro resistance of the sealed teeth to microleakage (p<0.001).The distributions of microleakage scores for the acid etched (Group I) and sequentially lased and acid etched (Group II) teeth are listed in 9. The filler within the sealant was observed to remain homogeneously distributed when the material was placed in contact with the acid etched enamel surface; however, the enhanced surface roughness of the lased teeth caused the segregation and concentration of the filler particles at the enamel-sealant interface. Corresponding elemental carbon, barium, silicon, and aluminium EDX maps of the sealed Group I and II teeth are presented in Back-scattered electron images of the enamel-sealant interfaces of acid etched (Group I) and sequentially lased and acid etched (Group II) teeth are shown in ® hydro™ demonstrated that this sealant is a ductile material that is highly filled with uniformly distributed micrometre and nanometre sized silicon-, aluminium-, and barium-bearing mineral phases. This finding contradicts the information contained in the manufacturer’s safety data sheet that indicates that this sealant contains aluminium and titanium oxides and does not list constituent silicon- and barium-bearing mineral phases  reported for contemporary commercially available resin-based fissure sealants cured under similar experimental conditions® hydro™ (VHN ~24) was found to fall within the range reported for commercial resin-based sealants (VHN from ~9 to 75)11. In general, despite the reduced curing efficiency of highly filled resin-based sealants, microhardness tends to increase with increasing filler-content11. In this respect, submicron and nano-sized fillers, such as those present in this sealant, are regarded to be particularly effective in increasing microhardness and reducing plastic deformation under occlusal forces11.The microhardness of UltraSeal XT® hydro™ were found to be below the detection limits of both the EDX instrument and the ion-selective electrode. In contrast, during a 30-day period under similar experimental conditions, other commercially available resin-based fissure sealants are reported to release up to 58 μg cm-2 of free fluoride ions11. However, these reported levels of fluoride release are unlikely to be significant regarding any beneficial impact on acid- and caries-resistance of local enamel, and, in this respect, the lack of detectible fluoride release from UltraSeal XT® hydro™ is not regarded to be a comparative disadvantage.Any fluoride species present in UltraSeal XTin vitro resistance of the sealed teeth to microleakage (10 (2013) who investigated the impact of laser ablation as an adjunct to acid etching to precondition molars prior to sealing with a filled hydrophilic sealant. Other studies, however, report that lasing prior to acid etching provides no statistically significant improvement in resistance to microleakage5. This lack of consensus on the effectiveness of Er:YAG laser ablation prior to acid etching is likely to arise from the range of laser settings employed by various researchers and the suitability of different sealants to the lased enamel surfaces10.Data obtained under the selected experimental parameters in this study indicate that laser ablation prior to conventional acid etching significantly improves the oleakage . The sup13.The principal mode of adhesion of resin-based fissure sealants to enamel is via the formation of micromechanically interlocking tags that penetrate the macro- and micro-porosities presented by the preconditioned enamel. Very little physicochemical interaction exists between the high energy surface of the hydroxyapatite in enamel and the organic constituents of the sealant9. Potential advantages of laser ablation prior to acid etching are reported to be: enhanced roughness and wettability of the lased enamel surface; more effective removal of pellicle and debris; superior conditioning of aprismatic hydroxyapatite; and enhanced acid-resistance of lased enamel9. Disadvantages of laser conditioning include the potential vitrification of the enamel surface at subablative laser energies and excessive roughness and microcracking under extreme ablation12. Laser conditioning of enamel is a relatively recent technique and further research is required to optimise the operating parameters.Enamel preconditioning by etching with phosphoric acid gel is currently the most common method used to prepare the occlusal tooth surface to receive the fissure sealant. This method of etching may not prove entirely effective on aprismatic enamel located at the fissure entrance and in the presence of remnant pellicle biofilm and organic debris lodged in deep narrow fissures10. In addition, this study has demonstrated that inorganic filler particles within a sealant may partially separate out from the resin phase and congregate at the interface as it flows over the rough lased surface. It is not presently known whether this congregation of filler particles at the interface of lased enamel will have a detrimental impact on the long-term clinical performance of the sealant.It is conjectured that the more viscous fissure sealants may fail to flow readily and fully adapt to the rougher surfaces presented by lased enamelAt present, all commercial resin-based fissure sealants are designed for acid etched enamel. It is suggested that, to take full advantage of the potential benefits of laser conditioning, a new generation of fissure sealants, which are specifically tailored to adhere and adapt to lased enamel, is required.® hydro™ , and to investigate its in vitro resistance to microleakage after placement on conventionally acid etched and sequentially lased and acid etched molars.The purpose of this study was to characterise the new resin-based hydrophilic fissure sealant, UltraSeal XT® hydro™ comprises irregular submicron and nano-sized silicon-, barium-, and aluminium-bearing filler phases embedded in a ductile resin matrix. Under standard light curing, UltraSeal XT® hydro™ achieved a degree of conversion (50.6±2.2%) and a Vickers microhardness (24.2±1.5) towards the lower end of the reported ranges for other commercial acrylate-based fissure sealants.Scanning electron microscopy and energy dispersive X-ray analysis of fracture and polished surfaces indicated that UltraSeal XTLaser preconditioning was found to significantly decrease microleakage and to increase enamel surface roughness, which caused zoning of the filler particles at the enamel-sealant interface.


Solid tumours comprise, not only malignant cells but also a variety of stromal cells and extracellular matrix proteins. These components interact via an array of signalling pathways to create an adaptable network that may act to promote or suppress cancer progression. To date, the majority of anti-tumour chemotherapeutic agents have principally sought to target the cancer cell. Consequently, resistance develops because of clonal evolution, as a result of selection pressure during tumour expansion. The concept of activating or inhibiting other cell types within the tumour microenvironment is relatively novel and has the advantage of targeting cells which are genetically stable and less likely to develop resistance. This review outlines key players in the stromal tumour microenvironment and discusses potential targeting strategies that may offer therapeutic benefit.•Benchsidein vitro and animal studies support the concept of stromal-directed treatment.○ The tumour stroma consists of mesenchymal, immune and vascular cells housed in an extracellular matrix. Stromal cells and extracellular matrix proteins represent genetically stable targets which can be exploited in cancer treatment. Numerous •Bedside○ Several therapeutic strategies have been developed or repurposed to target the stroma. The anti-angiogenic agent bevacizumab was one of the first specific stromal-targeting agents to be licensed for cancer treatment over a decade ago. More recently, immune modulation of the stroma has become a hugely successful strategy, with novel drugs such as checkpoint inhibitors set to revolutionise cancer treatment.•Governments○ Funding bodies should continue to acknowledge the pivotal role that the stroma plays in cancer progression, in parallel with cancer cell itself. Undoubtedly, the most successful treatment regimens of the future will address both the “seed” and the “soil”. In normal physiology, myofibroblasts are capable of closing a wound. Cancer is considered to be a ‘wound that does not heal’ in vitroCAFs express the enzyme lysyl oxidase (LOX) and lysyl oxidase-like proteins (LOX-L) 1–4 which allow crosslinking of ECM substrates such as collagen with elastin. This stiffens the ECM and stimulates integrin-dependent mechanotransduction pathways which promote invasion CAFs also play an important role in angiogenesis by secreting fibroblast growth factor (FGF)-2 +cKit-) into the circulation. The activated bone marrow cells secrete granulins creating a desmoplastic myofibroblast-containing stroma around indolent responding tumour cells, stimulating their expansion +cKit− cells and or granulin secretion seems appealing. Unfortunately, this has not materialised into any human studies which directly target CAFs. As Bateman Bone marrow cells are important in determining CAF transdifferentiation and stromal histology. Systemic endocrine signals such as osteopontin from tumours recruit particular bone marrow cells  with a mean duration of 1 year. There were no treatment related deaths but the expected haematological toxicities were observed. However, the only patient who was EBV seronegative at recruitment became seropositive and developed a lymphoma 4 months after treatment. Importantly, persistence of CD8+ cells after transfusion determined the degree of efficacy in this study Tregs are CD4+/CD25+ T cells specifically expressing FoxP3 which are capable of suppressing the effects of other immune cells in vitro and in vivo in a TC-1 tumour model The PI3K–AKT pathway is an important regulator of Treg activity. Selective inhibition of the PI3Kδ isoform has been shown to repress AKT activation and proliferation of Tregs Immune checkpoints are important regulators of CD8+ and Treg cell activity. In the cancer setting, a vast number of new antigens are generated. To prevent autoimmunity, immune checkpoints are activated to dampen pro-inflammatory T cell responses, at the cost of allowing immune escape of cancer cells Th cell subtypes have antagonistic effects on cancer progression. Th1 cells activate cytotoxic CD8+ T cells and have been shown to mediate tumour regression in a murine pulmonary fibrosarcoma model highIL-10low) is associated with expression of pro-inflammatory cytokines and tumour rejection, whereas alternative M2 polarisation (IL-12lowIL-10high) is associated with tumour progression In terms of macrophages, classical M1 polarisation (IL-12InRG7155 (emactuzumab). vitro, this resulted in cell death of TAMs. In vivo, this led to reduction of TAMs with an associated increase in CD8+ T cells and was associated with less tumour growth in animal models of colorectal cancer and fibrosarcoma. In a small phase 1 study, administration of this agent effected at least a partial metabolic response in all 7 patients with diffuse-type giant cell tumour. More recently, Cassier et al. In order to selectively target TAMs, Luo et al. in vitro cancer models in vivo cytokine stimulation and adoptive transfer of ex vivo-stimulated autologous, allogenic or NK cell lines. Early phase 2 studies used IL-2 to stimulate resident NK cells and proved to be effective in metastatic melanoma ex vivo using IL-2. Autologous NK cells were then injected into the tumour cavity and/or intravenously in a total of 16 courses. MRI showed partial responses after 3 of the 16 courses. Importantly, there were no significant neurological side effects. Another phase 1 study in patients with NSCLC used allogenic NK cells from donor relatives, expanded ex vivo with IL-15 and hydrocortisone. There was partial response in 2 of 16 patients and disease stabilisation in 6 patients. Again, there were no major local or systemic side effects NK cells are innate to the immune system and are able to directly kill tumour cells in several different ex vivo. The MART-1 T cells were then given to 9 patients with advanced melanoma in a total of 17 infusions. This therapy has the benefit of not requiring lymphodepletion or IL-2 treatment and consequently there were no severe adverse effects. One patient had a complete metabolic response which lasted 54 months and 4 others had stabilisation of disease to at least day 70.APCs process foreign antigens and present them alongside MHC class 1 or 2 molecules to naïve CD8+ and Th cells respectively. Professional APCs such as dendritic cells, macrophages and B cells are so named because they process and present antigens most effectively 4The tumour vasculature consists of ECs and pericytes. ECs form stromal capillaries and pericytes provide structural support. In healthy tissue, pericytes intimately cover ECs and through the expression of VEGF and angiopoetin-1 they lead to increased EC survival and structural stabilisation It is well established that hypoxia develops as a tumour expands and that its size is limited without neovascularisation or angiogenesis. Folkman et al. in vivo model. Moreover, VEGF inhibition has mostly had clinical success in combination with traditional chemotherapy, possibly because it normalises stromal vessels and allows better drug delivery Several monoclonal antibodies have been developed to target VEGF-driven angiogensis. Bevacizumab, targeting VEGF-A, is the most well known amongst these. It received US FDA approval in 2004 for use in metastatic colorectal cancer in combination with standard chemotherapy Another class of anti-angiogenic drugs are the VEGF/PDGF-receptor tyrosine kinase inhibitors which have shown significant response in several phase 3 trials. These drugs inhibit tyrosine kinase receptors from activating intracellular serine/threonine kinases such as Raf, resulting in reduced proliferation and angiogenesis Whereas anti-angiogenic drugs target new vessel formation, vascular damaging agents (VDAs) target existing vessels, causing ischaemia and haemorrhagic necrosis of the tumour An interesting ligand based approach is to fuse toxins to stromal vascular ligands. Rosenblum's group constructed the fusion molecule VEGF(121)/rGel which combines a VEGF ligand with the plant toxin gelonin Reduced oxygen tension in the TME leads to upregulation of hypoxia inducible factors (HIFs) by ECs 5The ECM is comprised of proteoglycans such as hyaluronan and versican and fibrous proteins such as collagen, elastin, fibronectin, laminin, periostin and tenascin-C Hyaluronan is associated with a permissive TME In vitro, periostin was shown to directly increase proliferation of several colorectal cancer cell lines. This effect was attenuated by addition of a periostin-specific antibody which triggered cancer cell apoptosis and worked synergistically with 5FU. Animal studies have shown that MZ-1, a monoclonal antibody to periostin, can reduce growth and metastatic potential of A2780 ovarian cancer xenografts The matricellular protein periostin is a ligand for αvβ3 and αvβ5 integrins on epithelial cells, promoting cell motility In vivo, decorin transfected xenografts were smaller and showed less neovascularisation. Recently, Xu et al. Decorin was shown to be differentially expressed in the tumour mass of malignant angiosarcomas compared to benign haemangiomas Tenascin-C is preferentially expressed by various tumours 6MiRs are small (20–30 nucleotide) non-coding RNAs which repress protein translation by binding to the 3’ UTR region of mRNAs There are several observational studies assessing miR profiles in different malignancies. One systematic review of over 40 studies highlighted total  tumoral miR-21 overexpression and let-7 downregulation as key determinants of patient outcome In terms of miR-based cancer therapy, the miR-34a mimic MRX34 is currently in a phase 1 trial for patients with unresectable, advanced or metastatic hepatocellular carcinoma which is due to complete in 2016 (NCT01829971). MiR-34 is a tumour suppressor which is thought to oppose proliferation, migration and chemoresistance 7For a long time, Paget's theories about the “soil” remained in the shade and ignored. In recent years however, there has been a focus of research efforts in this field, simultaneously bringing to light a variety of stromal-directed therapeutic strategies. The great appeal of the stromal TME is its genetic stability and reduced likelihood of Darwinian emergence of resistance, as seen in cancer cells. Moreover, stromal-directed therapy offers two key benefits: firstly, it creates an arid “soil” making it more difficult for a tumour to establish at both primary and secondary sites; consequently, it reduces the required doses of traditional cytotoxic chemotherapeutic drugs. With this in mind, the future of cancer therapy looks promising. The ultimate treatment regimens will include interventions which manipulate each component of the TME, in both stromal and cancer compartments, to promote tumour rejection. Ideally, these regimens will be personalised for each individual depending on the cellular and molecular fingerprint of their TME. This is not unrealistic if we consider that adoptive immune cell therapy, which epitomises personalised cancer treatment, is already well established.In this review, we have highlighted key cellular and molecular targets within the stromal TME and summarised relevant preclinical and clinical data associated with each. The article provides up to date, structured and comprehensible information on the translational aspects of stromal cancer therapy and how it has evolved from bench to bedside.•The TME is a functional ecosystem of cancer cells and stroma which interact through an array of signalling molecules. The stroma consists of mesenchymal, immune and vascular cells together with the ECM.•In comparison to cancer cells the stroma is genetically stable and therefore, anti-stromal therapies are less likely to succumb to chemoresistance.•There is huge excitement about redirecting and modulating the immune stroma to reject a tumour with the most successful approaches being adoptive T cell therapy and immune checkpoint inhibition respectively.•Targeting the vascular stroma with anti-VEGF agents is associated with intrinsic resistance. This has accelerated FDA approval of alternative anti-angiogenic agents such as sorafenib and prompted the development of mechanistically different drugs (VDAs).•The mesenchymal stroma and ECM are equally important in cancer progression but therapies specifically targeting CAFs, MSCs and ECM proteins are fewer and in comparatively earlier stages of development.•MiRs are master controllers of gene expression which are deregulated in cancer. MiR profiling has been shown to be useful in cancer prognostication. MiR modulating therapies are now in early human trials.This is a review article and does not refer to any unpublished human or animal studies which we have undertaken.RB and AM conceptualised the article. RB, AS and AM designed the structure of the article. RB, MB, AS and AM drafted the article with RG, HA, JP and GT revising critically for intellectual content. All authors have approved the final article.Cancer Research UK (CRUK) Clinical Research Training Fellowship and a CRUK pre-doctoral bursary (A20526). CRUK made no direct contribution to study design; collection, analysis and interpretation of data; in the writing of the manuscript; or in the decision to submit the article for publication.RB is funded by a None declared.


H. pylori 26695 has provided a wealth of information enabling us to carry out in silico studies to identify new molecular targets for pharmaceutical treatment. In order to construe the structural and functional information of complete proteome, use of computational methods are more relevant since these methods are reliable and provide a solution to the time consuming and expensive experimental methods. Out of 1590 predicted protein coding genes in H. pylori, experimentally determined structures are available for only 145 proteins in the PDB. In the absence of experimental structures, computational studies on the three dimensional (3D) structural organization would help in deciphering the protein fold, structure and active site. Functional annotation of each protein was carried out based on structural fold and binding site based ligand association. Most of these proteins are uncharacterized in this proteome and through our annotation pipeline we were able to annotate most of them. We could assign structural folds to 464 uncharacterized proteins from an initial list of 557 sequences. Of the 1195 known structural folds present in the SCOP database, 411  are observed in the whole H. pylori 26695 proteome, with greater inclination for domains belonging to α/β class (36.63%). Top folds include P-loop containing nucleoside triphosphate hydrolases (22.6%), TIM barrel (16.7%), transmembrane helix hairpin (16.05%), alpha-alpha superhelix (11.1%) and S-adenosyl-L-methionine-dependent methyltransferases (10.7%).The availability of complete genome sequences of  Helicobacter pylori is a spiral shaped, anaerophilic, gram negative and sluggish growing flagellated human pathogen existing in the gastric mucosa of human stomach. This bacterium was identified in 1982 by Barry Marshall and Robin Warren H. pylori infection, probably reaching up to 70% in developing countries and relatively less in industrialized countries (20–30%) according to World Gastroenterology Organisation [http://www.worldgastroenterology.org/assets/downloads/en/pdf-/guidelines/11_helicobacter_pylori_developing_countries_en.pdf]. In most of the cases, H. pylori infections are lingering but asymptomatic, majority of the patients never experience symptoms or some may have only mild gastric inflammation, acute infection may cause clinically relevant chronic active gastritis, peptic ulceration H. pylori possess significant genotypic diversity which engenders various strategies to interact with host cells, manipulate their behaviors in order to survive and propagate. Disease outcome is greatly influenced by bacterial genotype, host- physiology, genotype, dietary habits H. pylori has the ability to tolerate acidic conditions in the gastric environment by creating a positive inner-membrane potential at low pH H. pylori infections are centered on the use of a proton-pump inhibitors and antibiotics such as metronidazole and clarithromycin H. pylori strain 26695 consists of 1,667,867 base pairs with 1,590 predicted coding sequences and has well-built systems for motility, scavenging iron, DNA restriction and modification H. pylori 26695. The annotation of this genome/proteome would help in identifying better drug targets that can be exploited to tackle H. pylori infections, and it is one of the ultimate goals of this sequencing project. Often, functional annotation of a proteome is achieved from comparative sequence analysis. These methods have limitations since they mainly rely on the comparisons based on sequence homology. As a result, all proteomes sequenced so far have ∼40–60% unannotated proteins Genome sequence analysis revealed that the circular genome of ab-initio modelling methods can also be employed. From the validated 3D structures, types of folds and the active site can be characterized. The 3D structures of 145 proteins in H. pylori are so far determined experimentally and deposited in protein structure databank (PDB) H. pylori proteome.Focus on three dimensional (3D) structural organizations of proteins would help in deciphering the protein fold, structure and active site, which have applications in the structure based drug design methods that are rational. The availability of complete genome sequences has provided a platform to decipher the structural and functional information of any complete proteome using the computational methods. The results are reliable and provide a solution to the time consuming and expensive experimental methods. The information about function of a protein resides in its structure; the high resolution 3D structures of proteins are determined using X-ray crystallography and NMR techniques. In the absence of experimental structures, sequence homology methods are employed based on the understanding that proteins which share sequence similarity would also have homologous structure and function, barring a few examples http://genolist.pasteur.fr/PyloriGene) proteins using different sources in various sequential steps, followed by structure validation. The theoretical models are further subjected to analysis as a way to gain insight into their function. Functional annotation has been assigned through fold to function association as well as by the identification of ligand binding sites and cavities associated with that model. Fold prediction methods attempt to detect structural folds that are compatible with a particular query sequence based on similarities between query protein sequence and proteins of known 3D structure. Since protein surface dictates the type of interaction it can make with its associated ligand or other interacting partners, we further analyzed the protein structures through their binding sites. The overall objective is to predict as accurately as possible the probable function of the protein, at sequence and structure level. At amino acid sequence level we have annotated the protein by gene ontology to decipher the function. At the structure level we assigned structural classification, fold, ligand location (binding site) and ligand type  based on the template structure. The flow chart shown in H. pylori 26695 proteome.In order to understand the biological role of large numbers of linear amino acid sequence data generated through genome sequencing projects, we need to have knowledge of their structure. Even though structures determined by experimental methods provide high-resolution data, due to various limitations, structures cannot be determined experimentally for a large proportion of these sequences. Computational structure prediction techniques provide significant and reliable information, and are cost effective as well as less time consuming. Our approach started with finding structural models of the individual Pylorigene database  is a database containing comparative protein structure models of various organisms and relies mainly on MODELLER ab initio folding and threading methods. Information about all the structures with their respective source are provided in ModBase https://sites.google.com/site/lgpscuh/links.Quality of all the protein models obtained through ModBase, Phyre2 and I-TASSER were analyzed using Verify-3D ab initio approaches, were further analyzed by PSIPRED which is an accurate secondary structure prediction method that incorporates two feed-forward neural networks and performs an analysis on the output obtained from PSI-BLAST. Here we have used pGenTHREADER method Further the proteins where good quality structures could not be built either by homology modelling or The PDB structures and the validated protein model structures of good quality obtained through ModBase, Phyre2, and I-TASSER along with the associated target structures identified by PSIPRED and FUGUE were submitted to 3D-BLAST Ligand binding is a key aspect of protein function, mediating the ability of proteins to recognize their natural ligands for transport, signal transduction, catalysis and etc. This information also aids in the modulation of their function through the discovery of inhibitors. In COFACTOR method H. pylori proteome consists of large number of weakly annotated proteins termed as ‘predicted’ since they are largely derived through sequence comparisons. The database also has list of proteins which have not been studied in H. pylori but homologs have been characterized in other organism or gene has not been studied in any other organisms and hence are annotated as ‘predicted coding region’ and ‘predicted coding regions with no homologs in database’ respectively. Most of these proteins are uncharacterized in Pylorigene database, but through our annotation pipeline we were able to annotate most of them. Previously uncharacterized proteins (557) were listed in the database, and through this methodology adopted by us, we could annotate 464 proteins as shown in H. pylori database. Recently, Resende et. al., 2013 H. pylori 26695 using merlin software tools and on-line databases. EC numbers and TC numbers to metabolic gene encoding enzymes and transport proteins, respectively have been assigned. In spite of differences in the methodologies, we found that our functional annotation of nearly 95% proteome is agreeable with that of Resende et al., 2013.Pylorigene database, an annotated H. pylori complete proteome can be useful in analyzing the fold content and fold preferences of this organism which will be further helpful in identifying important folds that are sufficient for sustaining life and causing infection to the host. Also, the knowledge of structural fold of a protein important in causing disease could be a good target for structure based drug design studies. Assignment of structural folds in the whole proteome is performed through the SCOP database. The fold analysis carried out in this proteome evidently shows the existence of all seven major structural classes (as per SCOP classification) as indicated in The availability of the structural models covering H. pylori proteome, with greater inclination for domains belonging to α/β class (36.63%). The top folds  are observed in the whole op folds  in the min silico results. In the absence of such data, 3D structures were either built or at least the fold was predicted. The assessment of each model was made through different estimates of confidence, for example, statistical significance of alignments, extent of sequence similarity, geometry and stereochemistry when compared to high resolution crystal structures, and primary sequence to 3D structure correlation. We therefore believe that the results from these methods are highly reliable and reproducible.A significant feature of this annotation pipeline involved obtaining structures from databases of experimental and First example we discuss in this category is HP0773  that is labeled as predicted coding region HP0773 in Pylorigene and UniProt databases. Our analyses described the protein as nitroalkane dioxygenase that is also known as nitroalkane oxidase (NAO). NAO is a structural member of the flavoenzyme acyl-CoA dehydrogenase (ACAD) superfamily. These enzymes are mainly involved in catalyzing oxidative denitrification of neutral nitroalkanes to their equivalent carbonyl compounds like aldehydes or ketones, hydrogen peroxide and nitrite using FAD or FMN as a cofactor. These nitroalkanes are further used as intermediates for synthesis in chemical industries 8 barrel domain. The COFACTOR server 8-barrel proteins).NAO comprises of two domains, a triose phosphate isomerase (TIM) barrel domain and C- terminal domain with a novel folding pattern (αααβαβα fold). The TIM barrel domain has eight parallel β strands and eight α-helices. Between these two domains a cleft is located where flavin mononucleotide (FMN) is bound as a cofactor. In the deep binding pocket situated near the boundary of two domains, one molecule of non-covalently bound FMN is present and majority of the binding site residues are contributed by the main (β/α)The edge of isoalloxazine ring of FMN is somewhat accessible from the protein surface. The FMN binding pocket within 5 Å region is lined by amino acid residues, model (template), Gly22 (Gly21), Gly23 (Gly22), Met24 (Met23), Gly25 (Gln24), Val26, Asn99 (Asn73), Leu101 (Thr75), Ile147 (Lys124), Glu175 (Asp145), Ser179 (Cys149), Gly180 (Ala150), Gly181, His182 (His152), Ala219 (Ser178), Gly220 (Gly179), Gly221 (Gly180), Gln240 (Asn199), Met241 (Met200), Ala242 (Gly201), Thr243 (Thr202), Leu246 (Leu205), Tyr338 (Tyr277), Phe339 (Ser288) and Asn343  as shown in Further oxidation of the neutral nitroalkanes by NAO needs a catalytic base to initiate oxidation of the neutral substrates by abstracting a proton from the substrate α-carbon Thermus thermophiles. Both model and template structures superimposed with low RMSD (0.09 Å)  which is an uncharacterized protein in the database and we annotated it as uracil phosphoribosyltransferases (PRTase). This protein was modelled by Phyre2 server using PDB_ID: 1WD5_A as template which corresponds to crystal structure of a predicted phosphoribosyltransferase from (0.09 Å) . AccordiSulfolobus solfataricus (PDB_ID: 3G6W), Aquifex aeolicus (PDB_ID: 2E55) and Bacillus caldolyticus (PDB_ID: 1I5E). The COFACTOR server predicted inhibitor bound crystal structure PDB_ID: 3G6W as template which has similar binding site as that of model. Analysis of ligand binding residues showed that these regions overlap appreciably well with the modelled protein as shown in The Dali homology search of the model showed close similarity with uracil phosphoribosyltransferase from various organisms like de novo pathways, from unrelated compounds as well as by salvage pathways by converting the preformed bases and nucleosides to nucleotides. PRTases catalyze the reversible transfer of a phosphoribosyl group from 5-phosphoribosyl-α-1-diphosphate (PRPP) to N1 nitrogen of base resulting in the formation of β-N-riboside monophosphate. The product β-N-riboside monophosphate formed is specified by the base present which can be adenine, guanine, hypoxanthine, xanthine and uracil. Further these PRTs are classified into type I and type II based on the presence and absence of a 13 amino acid sequence (PRPP binding motif), respectively Purine and pyrimidine nucleotides can be synthesized both by The salvage enzyme uracil phosphoribosyltransferase (UPRTase) belonging to the type I family of PRTases, catalyzes the conversion of uracil and PRPP to uridine monophosphate (UMP) and diphosphate (PPi) The COFACTOR server predicted that PRPP binding pocket is enclosed by model (template) Leu51 (Ile78), Ser52 (Leu79), Phe53 (Arg80), Asn54 (Ala81), Asp151 (Asp140), Arg152 (Pro141), Gly153 (Met142), Ile154 (Ile143), Glu155 (Ala144), Thr156 (Thr145), Gly157 (Ala146), Phe158 (Ser147) and Arg159 (Thr148) as shown in Listeria monocytogenes. Both model and template structures superimpose with low RMSD (0.13 Å)  which is unknown in the Pylorigene database but we annotated it as methyltransferase. This protein was modelled by Phyre2 using PDB_ID: 3LPM as template which is the crystal structure of putative methyltransferase small domain protein 2 from (0.13 Å) . ModelleMethyltransferases form a large group of enzymes that methylate a variety of substrates but can be segregated into several subclasses based on their structural features. The most common class of methyltransferases is class I that contains a Rossman fold for binding S-adenosyl methionine (SAM) while class II methyltransferases have a SET domain. Methylation of proteins is found to have regulatory role in protein activation, protein-protein interactions and protein-DNA interactions. They also play an important role in methylation of ribosomal RNA (rRNA) nucleotides, which further helps in the biogenesis and activity regulation of the ribosome, such as fine-tuning of local rRNA structure, 30S subunit assembly and antibiotic resistance Escherichia coli (PDB_ID: 2B3T), Escherichia coli K-12 (PDB_ID: 4DCM), Pyrococcus horikoshii (PDB_ID: 1WY7) and Thermus thermophiles (PDB_ID: 3CJT).The Dali homology search of the model structure showed close resemblance with RNA methyltransferase from various organisms such as Ligand binding site prediction made on the model structure using COFACTOR server identified a pocket that overlaps with the inhibitor bound crystal structure of 16S rRNA methyltransferase belonging to class I of methyltransferases (PDB_ID: 2ZWV_A) which is co-crystallized with ligand SAH (S-adenosyl-L-homocysteine). These sites are also detected by the pocket predictions by the CASTp server as shown in NADPH-dependent 7-cyano-7-deazaguanine reductase).The proteins for which reliable and validated model structures could not be built, were further analyzed with high confidence using fold-based prediction methods (PSIPRED and FUGUE) and useful annotation could be attributed to some unknown proteins. Some of these examples include, HP0013 (tRNA (5-methylaminomethyl-2-thiouridylate)-methyltransferase), HP0031 , HP0728 (isoleucyl-tRNA lysidine synthetase), HP1211  and HP1413 , HP0130 (DNA binding), HP0158 (N-linked glycosylation), HP0980 , HP0971 (transport protein) and HP0935 (N-acetyltransferase protein).H. pylori 26695 proteome enhances our knowledge about this pathogenic organism and further provides guidance to find new drug targets.This systematic structural and functional annotation of H. pylori 26695 is not feasible since there are several hundreds of proteins which have to be biologically or biochemically characterized. This current work provides useful information for better understanding of H. pylori 26695 strain proteome by providing information on the 3D structure of a protein, which is further useful to predict the structure-function association and cellular functions. The structural annotation reported here covers a significant proportion of the H. pylori proteome. This annotation has provided insights about the folds for a significant number of proteins and importantly indicate that cellular metabolism in H. pylori 26695 can be achieved with only 411 folds and their various combinations. High confidence molecular models can now be obtained for several proteins, which along with the experimental structures available for that species can provide a first glimpse of the structural proteome as well as key residues and motifs present in the functional sites of that protein.In recent years, the need to design and develop novel antibacterial agents has become crucial due to the global outbreak of infectious diseases. Now and in the immediate future, the understanding of the functions of all proteins in H. pylori can be readily applied for other organisms as well and will fill the ‘blank spots’ in respective proteomes. At a later stage, the new drug targets can be exploited to identify novel inhibitors using computational methods, as recently demonstrated for H. pylori DapE Assigning of structure-function to several unknown proteins that may be probable virulence determinants will allow critical tests of their functions, cellular targets as well as the innate and adaptive immune responses of the host. This will further aid in novel understanding into their mechanisms of early colonization, persistence of this bacterium during long-term carriage, and the mechanisms by which it promotes various gastroduodenal diseases, and are therefore novel drug targets. The large scale annotation pipeline used here to derive biological insights about S1 TableTable showing quality estimation values from Verify_3D and PROCHECK for each protein with their respective source.(DOC)Click here for additional data file.S2 TablePgenthreader results for the failed protein models showing template PDB_ID, confidence and P-value.(DOC)Click here for additional data file.S3 TableFUGUE results for the failed protein models mentioning template PDB_ID, confidence and Z-score.(DOC)Click here for additional data file.S4 TableDALI results for the passed protein models mentioning Z-score, template PDB_ID and sequence identity.(DOCX)Click here for additional data file.S5 TableDistribution of various SCOP-ID, class, fold, superfamily and family across whole proteome.(DOC)Click here for additional data file.S6 TableComparison of previous annotation from Pylorigene database and new annotation from our work.(DOC)Click here for additional data file.