Thirdhand smoke (THS) is the fraction of cigarette smoke that persists in indoor environments after smoking. We investigated the effects of neonatal and adult THS exposure on bodyweight and blood cell populations in C57BL/6 J mice. At the end of neonatal exposure, THS-treated male and female mice had significantly lower bodyweight than their respective control mice. However, five weeks after neonatal exposure ended, THS-treated mice weighed the same as controls. In contrast, adult THS exposure did not change bodyweight of mice. On the other hand, both neonatal and adult THS exposure had profound effects on the hematopoietic system. Fourteen weeks after neonatal THS exposure ended, eosinophil number and platelet volume were significantly higher, while hematocrit, mean cell volume, and platelet counts were significantly lower compared to control. Similarly, adult THS exposure also decreased platelet counts and increased neutrophil counts. Moreover, both neonatal and adult THS exposure caused a significant increase in percentage of B-cells and significantly decreased percentage of myeloid cells. Our results demonstrate that neonatal THS exposure decreases bodyweight and that THS exposure induces persistent changes in the hematopoietic system independent of age at exposure. These results also suggest that THS exposure may have adverse effects on human health. THS is residual tobacco smoke that clings to indoor surfaces, and re-emission of gases and resuspension of particles from contaminated surface materials after active smoking has ceasedThe toxicological and adverse health impacts of active smoking and secondhand smoke (SHS) have been extensively studied before. However, the process of characterizing the potential biological and health effects of THS is just beginning. Chemical analyses of THS composition reveal that THS contains many classes of toxic compounds, including both semi-volatile (SVOCs) and volatile organic compounds (VOCs), as well as chemicals with incomplete toxicity testing23456et al. demonstrated for the first time that exposure to laboratory-generated THS causes significant DNA damage in human cell lineset al. showed cytotoxicity of THS in both mouse and human cell lineset al. reported animal studies showing that THS exposure resulted in damage to multiple organs and behavioral alterations in miceet al. showed that THS exposure increases the risk of thrombosis-based disease stateset al. found that the exposure to THS at very low concentrations caused distinct metabolic changes in two different types of male reproductive cell linesOxidants in cigarette smoke can cause local and systemic inflammation. Recently, Hang et al. found that the calculated cancer risk for children (1 to 6 years old) is increasedSmall children are a particularly vulnerable population who are exposed to THS toxicants through inhalation, ingestion and dermal contact. By analyzing nicotine and nitrosamines/TSNAs in house samples, Ramirez The link between SHS exposure and body weight and immunological parameters has been investigated extensively in the past1415162 swatches of THS-exposed 100% cotton terry cloth in their cages for 3 weeks. THS exposure cages contained standard bedding material plus THS cloth, while control cages contained standard bedding only  is a standard test to assess general health status. Therefore, we used a 20-parameter CBC to investigate the effects of THS exposure on the abundance of different cell populations in mouse blood. When tested at 17 weeks, both male and female mice in the neonatal THS exposure cohort had almost 3 times as many eosinophils (EO) and correspondingly higher EO percentages than controls . Some seApparently, red blood cells (RBC) were not affected, as both RBC counts and hemoglobin (HB) levels were not significantly altered. However, we found that platelet counts were significantly lower in neonatal-exposed male and female mice and adult-exposed male mice . AdditioAlthough there was no difference in absolute blood lymphocyte numbers between THS-exposed and control mice, further analysis by FACS (fluorescence-activated cell sorting) revealed significant changes in lymphocyte subpopulations. Because of children’s exploratory behavior and metabolic properties, it is likely that they are at increased health risk when exposed to THS toxicants. For example, children who spend a lot of time on THS-laden carpets could breathe in a significantly larger amount of dust-bound pollutants than adults, while their skin is in near-constant contact with carpet or other furniture surfaces. Dust ingestion was identified as the dominant  source of exposure to indoor semivolatile environmental pollutants in toddlersThe most striking finding of this study was a significant reduction in weight gain in both THS-treated male and female mice exposed to THS from birth until weaning at 3 weeks of age. Similarly exposed adult mice did not show THS exposure induced reduction in bodyweight. After the neonatal exposure to THS ended, the bodyweight of exposed mice caught up to control mice at 5, 8, 12 and 17 weeks. To our best knowledge, this is the first report of bodyweight change caused by exposure to THS under conditions that mimic human exposure. The links between active cigarette smoking, smoking cessation and bodyweight change have been investigated extensively in the past. In some previous studies it has been shown that adult smokers lose weight as compared to non-smokers, and tend to gain weight after they quit smokingAlthough the exact mechanisms underlying the changes in weight gain we observed are still unclear, tobacco use is known to be associated with appetite suppression and nicotine is considered an appetite suppressant. Moreover, nicotine has been reported to induce bodyweight loss in both human and rodent studies through mechanisms including affecting hormones that control caloric intake and fat metabolisms202122237There is a link between active and passive smoking and changes in complete blood counts, which may underlie the pathophysiological mechanism of the biological effects of tobacco smoking2627We further studied the effect of neonatal exposure to THS on specific immune subpopulations in mouse blood. As shown in In conclusion, our data show that THS exposure can inhibit normal weight gain in neonatal mice. We have also shown that THS exposure, during infancy and during early adulthood, changes the circulating populations of blood cells from both the myeloid and lymphoid lineages. Our research also shows that neonates are more vulnerable to potential health effects caused by THS exposure than adults. Our findings provide further evidence that THS exposure can have biological effects and supports nonsmoking policies as the best way to protect nonsmokers from the health effects of tobacco smoke exposure.3 stainless steel chamber for a total of 550 hours over 1,190 days. The smoke exposure chamber was ventilated for approximately 550 hours during this time. During smoking, a total of 5,600 mg of particulate material was introduced into the steel chamber. This is equivalent to the smoke from 400–700 cigarettes over 3 years and 4 months, or approximately 1/3–2/3 of a cigarette per day.The THS samples were generated in a laboratory system to simulate chronic THS exposure, as described in the previous study2 swatches) of THS-exposed cloth per cage, with a nicotine loading of 20 μg/g. Assuming that uptake of nicotine through ingestion, inhalation and dermal routes was quantitative, the predicted dose was 50 μg/day/kg of bodyweight. This value is comparable to the ingestion exposure of a toddler estimated by Bahl et al.C57BL/6 mice were divided into experimental and control groups. The neonatal experimental group was exposed to THS from birth for 3 weeks; the control group was never exposed to THS and was housed separately from the THS-exposed mice. The adult experimental group was exposed to THS from 12 to 15 weeks of age. All mice were fed a standard chow diet . Mice were bedded on the THS-laden cloths along with standard bedding in mouse cages and then switched to standard bedding only. The cloth swatches were changed once a week with standard cage change. The cloth was the sole source of smoke exposure. Possible routes of THS exposure include inhalation, ingestion and dermal absorption. There was 0.85 g . A complete blood cell count was acquired using a HemaVet950FS and specific lymphocyte subpopulations were assessed by FACS with cell specific markers for B-cells, T-cells, T-helper and T-suppressor cells at 17-weeks using the following antibodies (BD Biosciences): rat anti mouse CD3-PE; rat anti mouse CC45R/B220 PerCP; rat anti mouse CD8a antibody APC; rat anti mouse CD4 antibody Alexa 488. The percentages of cells in blood were determined on BD FACS Calibur (Becton Dickinson) and data were analyzed with FlowJo software .The differences in bodyweight, the cellular components of blood and lymphocyte subpopulations between THS-treated and control group were assessed by Mann-Whitney test. Results with p-value < 0.05 were judged to be significant. Statistical analysis was performed using SPSS version 12.0 .How to cite this article: Hang, B. et al. Early exposure to thirdhand cigarette smoke affects body mass and the development of immunity in mice. Sci. Rep.7, 41915; doi: 10.1038/srep41915 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Streptomyces sister-taxa which share a recent common ancestor and nearly identical small subunit (SSU) rRNA gene sequences, but inhabit distinct geographic ranges demarcated by latitude and have sufficient genomic divergence to represent distinct species. Here, we explore the evolutionary dynamics of secondary metabolite biosynthetic gene clusters (SMGCs) following lineage divergence of these sister-taxa. These sister-taxa strains contained 310 distinct SMGCs belonging to 22 different gene cluster classes. While there was broad conservation of these 22 gene cluster classes among the genomes analyzed, each individual genome harbored a different number of gene clusters within each class. A total of nine SMGCs were conserved across nearly all strains, but the majority (57%) of SMGCs were strain-specific. We show that while each individual genome has a unique combination of SMGCs, this diversity displays lineage-level modularity. Overall, the northern-derived (NDR) clade had more SMGCs than the southern-derived (SDR) clade . This difference in SMGC content corresponded with differences in the number of predicted open reading frames (ORFs) per genome  such that the ratio of SMGC:ORF did not differ between sister-taxa genomes. We show that changes in SMGC diversity between the sister-taxa were driven primarily by gene acquisition and deletion events, and these changes were associated with an overall change in genome size which accompanied lineage divergence.We have identified  Microbial secondary metabolism encapsulates a remarkable diversity of natural products with an extensive range of biological activities. Secondary metabolites differ from primary metabolites in that they are not involved in essential catabolic and anabolic activities required for normal growth and reproduction, but may contribute significantly to an individual’s fitness . While pThe values of natural products to humanity are widely recognized, yet because most research has focused on their discovery and human-centric relevance, we are still far from understanding their biological role in natural systems. The discovery and application of antibiotics revolutionized medicine in the 1940’s, sparking the “golden age” of antibiotics between 1950 and 1960, during which time approximately half of the microbial-derived drugs we use today were discovered . In addiStreptomyces [Streptomyces life cycle resembles that of many fungi, consisting of filamentous growth, formation of mycelia, and production of aerial hyphae and spores. Indeed, Streptomyces were thought to be an intermediary between bacteria and fungi until as recently as the 1950’s [Streptomyces are Gram-positive Actinobacteria with long linear chromosomes that have a high G+C content [Streptomyces species are often known to produce several secondary metabolites when grown in culture. Genome sequencing, however, reveals that Streptomyces contain an enormous reservoir of “cryptic secondary metabolites” which are not expressed under standard laboratory conditions [Streptomyces coelicolor A3(2) was known to produce several well characterized secondary metabolites, genome sequencing discovered that it actually contained >20 biosynthetic gene clusters not expressed when grown in culture [Soil-dwelling actinomycetes are the predominant source of microbial-derived therapeutic natural products, and the majority of described bioactive compounds originate from the genus ptomyces ,7. The Se 1950’s . However content . Traditinditions . For ins culture . Genes w culture . Phyloge culture ,17,18.Streptomyces isolated from prairie soils is highly variable between strains and correlates poorly with 16S rRNA gene phylogeny, suggesting a role of selection acting at small spatial scales [Streptomyces SMGC composition varies in relation to both spatial distance and environmental dissimilarity [Streptomyces for endemism at inter-continental and regional geographic scales [The evolutionary and ecological processes that govern SMGC diversity remain largely unexplored. The richness of SMGCs within soils is linked to both edaphic and biotic factors ,20. For l scales ,22. Convmilarity . Furtherc scales ,25 suggeStreptomyces is an ideal model system to evaluate the influence of SMGC dynamics on patterns of diversification. We previously assembled a culture collection of Streptomyces from sites spanning the United States, and we observed evidence for dispersal limitation, as well as a latitudinal gradient of species riches and intraspecific nucleotide diversity [Streptomyces sister-taxa that have geographic ranges delimited by latitude and have patterns of gene flow and genomic diversity consistent with their diversification from a recent common ancestor [Streptomyces sister-taxa to explore SMGC evolutionary dynamics during the divergence of Streptomyces species.Microbial biogeography is readily explored with geographically explicit microbial culture collections (reviewed in ), and thiversity ,28. Fromancestor . Here, wStreptomyces representing sister-taxa and related strains. These strains were identified through a phylogenetic analysis of a Streptomyces culture collection [We used comparative genomics to analyze patterns of genomic diversity and SMGC content in 24 llection  generateStreptomyces griseus species cluster [Streptomyces griseus subsp. griseus NBRC 13350 (Assembled genomes are 7.5–9.1 Mb with a G+C content of 71.4–72.5% and 6776–8078 predicted open reading frames (ORFs) . The cor cluster  and sharRC 13350 .The NDR core genome is comprised of 4234 genes, and the SDR core genome is comprised of 4400 genes. The NDR and SDR clades share a recent phylogenetic ancestor and have nearly identical 16S rRNA genes (inter-lineage nucleotide dissimilarity of 0–0.21% between strains). Strains within each clade have a whole genome ANI value ranging from 95.6% to 99.9%, while the ANI between strains of NDR and SDR range from 92.6% to 93.3% . DistincStreptomyces sister-taxa. To assess the novelty of these SMGCs, we utilized antiSMASH’s downstream annotation pipeline, which annotates SMGCs based on similarity to genes and pathways present within the Minimum Information about a Biosynthetic Gene cluster (MIBiG) database. The antiSMASH pipeline annotated 120 SMGCs across the 24 strains . We used antiSMASH  to ident strains . Each ge strains . This raptomyces ,34,35,36n = 25) of the MIBiG-annotated SMGCs represent well-characterized biosynthetic gene clusters (in which ≥70% of the genes in a SMGC show similarity to genes within the most similar known cluster from the MIBiG database)  . In addiStreptomyces sister-taxa encompass 22 classes of natural products. Most of these classes, including bacteriocin, butyrolactones, ectoine, lantipeptide, melanin, non-ribosomal peptide synthases (NRPS), siderophore, polyketide synthases (PKS), and terpene gene clusters, are widely conserved at the genus level [Streptomyces genomes [t-test with Bonferrori correction, p < 0.002). Additionally, NDR genomes harbor linaridin gene clusters, which are entirely absent from SDR genomes . Hence, while it seems clear that gene acquisition is a major driver of SMGC biodiversity, the role of gene acquisition in driving lineage divergence remains unclear.The majority of SMGCs observed within the sister-clades occurred in only one or a few strains, and this suggests that gene acquisition is a major force that drives the diversity of SMGC pathways in ry SMGCs , and thiry SMGCs  may undery SMGCs . Overallry SMGCs . This rery SMGCs  and largWe also see evidence that NDR has undergone the deletion of SMGC-associated genes inherited from the common ancestor of NRD and SDR. For example, the SRO15-2005 lassopeptide gene cluster is conserved in SDR and found in INT but absent from NDR, suggesting that deletion of this lassopeptide accompanied NDR divergence . We alsoTaken together, these results suggest that the sister-clades are under different evolutionary pressures which drive dissimilarity in SMGC composition. NDR genomes have increased in size relative to their ancestors suggesting an overall increase in the rate of gene acquisition via horizontal gene exchange, and this increase in gene acquisition has resulted in an increase in strain-specific SMGC content in NDR. In addition, the presence of NDR-specific core SMGCs  indicates that some horizontally acquired SMGC have gone to fixation within NDR. At the same time, deletion events in NDR have pruned away SMGCs inherited from ancestral lineages . We hypothesize that these changes in SMGC content are likely to have effects on fitness which should act to reinforce lineage divergence either as a result of antagonism or niche differentiation.Streptomyces isolated from grassland soils (pH 3.9–7.3) sampled at 0–5 cm from sites across the United States [Streptomyces cultures were obtained from air-dried soils on glycerol-arginine agar (pH 8.7) containing antifungals as previously described [We built a culture collection of >1000 d States . Pure Stescribed . GenomicStreptomyces genomic sequencing libraries were prepped with the Nextera DNA Library Preparation Kit , and draft genomes were generated using the Illumina HiSeq2500 platform  and paired-end 2 × 100 bp reads at the Cornell University Biotechnology Resource Center (BRC). Quality control and assembly was performed with the A5 pipeline [pipeline , and genpipeline . Multiplpipeline , and tripipeline . Ortholopipeline  with MCLpipeline . Genome The phylogenetic relationship between genomes was reconstructed from DNA sequences of multiple whole genome alignments using maximum likelihood (ML) with the generalized time reversible nucleotide substitution model  with gamSecondary metabolite biosynthetic gene clusters (SMGC) were predicted and annotated using the online server antiSMASH 3.0 . We alsoStreptomyces species that recently diverged from a common ancestor. Our objective was to observe and explore the evolutionary dynamics of SMGCs that accompany evolutionary diversification and to assess SMGC conservation within and between closely related species. It is clear that gene gain and loss events drive major differences in SMGC composition, both within and between species. While both species share conserved core SMGCs, each clade has its own species-specific SMGC core, and the majority of SMGCs were strain-specific. This pattern indicates that these SMGCs, not present in shared ancestors, were acquired recently due to horizontal gene exchange. We used comparative genomics to examine SMGC diversity within strains of two closely related In addition, we observe that SMGCs that have been inherited from a shared ancestor can vary considerably in gene content, both due to the acquisition and deletion of individual genes within each gene cluster. We observe SMGC gain and loss dynamics that differ between clades and identify SMGC acquisition and deletion events that correspond to ancestral diversification events. These findings show that SMGC modification is associated with lineage divergence, though whether these changes cause or reinforce divergence directly or are an indirect product of evolutionary divergence remains to be seen. A limitation of the comparative genomics approach is that we cannot assess the ecological activity of a pathway from genome sequence data. It is possible that some  of the strain-specific pathways, if acquired by recent horizontal exchange, may be non-functional. It is also possible that changes in SMGC architecture and gene content could alter pathway functionality and that pathways deemed orthologous on the basis of genetic similarity may have different functions in different strains.Finally, we can conclude that, while strains within a species will share a core set of SMGCs, the number of accessory SMGC within a given species can be quite large, with each strain having its own repertoire of strain-specific SMGCs. Furthermore, the majority of these strain-specific SMGCs remain uncharacterized and lack similarity to SMGCs documented in public databases.


Surgical excision is recommended as first-line treatment but in cases of extensive disease can lead to unacceptable functional morbidity or cosmetic outcomes. Radiation therapy has been shown to be equally efficacious to surgery in the treatment of carcinoma in situ but its use has historically been limited due to concerns regarding toxicity. In this case report we present a novel therapeutic technique that may enable radiotherapy to be employed as a definitive treatment option in these challenging cases.Confluent squamous cell carcinoma in situ of his right hand previously treated with 5-fluorouracil presented with recurrent biopsy-proven confluent squamous cell carcinoma in situ of multiple surfaces of his right hand and digits with involvement of nail beds. To avoid extensive resection and possible amputation he was offered definitive external beam radiation therapy utilizing a water bath as a tissue-equivalent bolus material. This protocol enabled improved dose homogeneity to the target volume while minimizing acute toxicity. He experienced complete clinical resolution of the disease with only minimal acute edema and hyperpigmentation. Twenty months following treatment completion he remains disease-free with normal function and excellent cosmesis.A 75-year-old white man with a previous history of carcinoma in situ.Therapeutic radiation utilizing water as a tissue-equivalent bolus in this complicated case enabled definitive treatment of disease without compromising functional or cosmetic outcomes. Radiotherapy may therefore be an alternative and under-utilized approach to surgical excision in difficult-to-treat cases of carcinoma  If left untreated, 3 to 5% of these in situ lesions will ultimately progress to invasive SCC and one-third of these invasive cancers may metastasize [Skin cancer is the most commonly diagnosed cancer in the USA; over three million patients are diagnosed as having basal cell or squamous cell carcinoma (SCC) each year . Squamouastasize .Confluent and extensive CIS, particularly involving digits and nail bed, represents a rare yet significant therapeutic challenge. Surgical excision is recommended as first-line treatment but in cases of extensive disease can lead to unacceptable functional morbidity or cosmetic outcomes . ExternaHere we discuss a rare case of extensive CIS involving multiple surfaces of the hand with extension to several digits and nail beds. Definitive EBRT employing a water bath technique is presented as an effective modality in treating extensive CIS with excellent clinical, functional, and cosmetic outcomes.A 75-year-old white man with a previous history of CIS of his right hand treated with topical 5-fluorouracil (5-FU) presented with new extensive lesions of his right hand. A dermatological examination demonstrated a confluent erythematous plaque covering the ventral, lateral, and dorsal surfaces of his right hand involving the digits and the second through to fourth nail beds. Shave biopsies revealed atypical keratinocytes throughout the epidermal layer consistent with CIS.Recommended surgical options would have probably required extensive skin grafting or amputation. He was therefore offered definitive EBRT utilizing a water bath as a tissue-equivalent bolus. He was simulated via computed tomography (CT) with all visible disease delineated with wire. His hand was immersed in a water basin filled to 2 cm above the dorsal surface, with 1 cm of tissue-equivalent Superflab below the ventral surface Fig. . His firCIS can progress to invasive SCC and requires aggressive treatment. Most commonly seen on skin regions chronically exposed to ultraviolet light radiation such as the head and neck, CIS has also been described of the vulva, anus, palm, digit, and nail unit . CIS typA variety of treatment options are available for CIS, including surgical excision, Mohs surgery, cryotherapy, photodynamic therapy (PDT), and therapeutic radiation . While sConfluent disease greater than 3 cm is rare and presents a particular challenge in achieving disease control while also providing acceptable cosmetic and functional outcomes . Severalet al. [Mohs surgery has been considered the standard of care for large lesions of the hand and digits, but can lead to contractures, loss of the fingernail, or amputation . Until ret al.  utilizedThe case presented in this report is unusual due to the extent and confluence of the disease encompassing multiple surfaces of the hand and digits, the involvement of multiple nail beds, as well as its recurrent status refractory to topical 5-FU. We utilized a water bath with Superflab to function as a tissue-equivalent bolus to negate the skin-sparing properties of megavoltage photon irradiation. This technique enabled the production of a uniform surface dose for optimal target volume dosing on all surfaces while minimizing radiation toxicity . The tre


This is due to the fact that the embedded ceria nanoparticles have optical tri-valiant cerium ions, associated with formed oxygen vacancies, with a direct allowed bandgap around 3.5 eV. In addition, the impact of chemical crosslinking of the PVA on the fluorescence emission is studied in both cases of adding ceria nanoparticles in situ or of a post-synthesis addition via a spin-coating mechanism. Other optical and structural characteristics such as absorbance dispersion, direct bandgap, FTIR spectroscopy, and SEM analysis are presented. The synthesized optical nanocomposite could be helpful in different applications such as environmental monitoring and bioimaging.This paper introduces a new fluorescent nanocomposite of electrospun biodegradable nanofibers embedded with optical nanoparticles. In detail, this work introduces the fluorescence properties of PVA nanofibers generated by the electrospinning technique with embedded cerium oxide (ceria) nanoparticles. Under near-ultra violet excitation, the synthesized nanocomposite generates a visible fluorescent emission at 520 nm, varying its intensity peak according to the concentration of  Ceria nanoparticles can be in the form of either dry powder or colloidal particles in solution. However, there is a lack of research in forming the active ceria nanoparticles in a solid host.Cerium oxide nanoparticles (ceria NPs) attract great research and commercial interests due to their observable capability to capture radicals and dissolved oxygen ,2. Theses (Ce4+) . The for achieve . Therefoet al., it was showed that the ceria formed on nanofibers has a non-active form  [in situ or in post-synthesis treatment using the spin-coating method on crosslinked electrospun PVA nanofibers (NFs). This method shows promising results in optical sensor applications such as peroxide [In this paper, we have produced active ceria NPs in a nanofiber host. In a research work done by Yang +; CeO2) . However+; CeO2) . Regardi+; CeO2) . Then, t+; CeO2) ,12. In t+; CeO2) . The res+; CeO2) ,15,16. C+; CeO2) , the fluperoxide .in situ ceria nanoparticles technique and the deposited ceria nanoparticles within a crosslinked PVA nanofiber technique. The embedded in situ ceria nanoparticle concentration can be controlled in PVA electrospun nanofibers with less ceria agglomeration inside the fiber [In this presented work, two different methods are introduced to produce fluorescent electrospun nanofibers, which are electrospun PVA nanofibers created with the embedded he fiber .The new contribution is depositing ceria nanoparticles in a crosslinked electrospun PVA nanofiber surface. However, this technique shows some difficulty in controlling the ceria nanoparticle concentration due to the losses that happen during the deposition technique; also ceria nanoparticle agglomerations were difficult to control, leading the presence of florescence quenching mechanism compared to the first technique, though it can be used in some applications such as drug delivery that deal with the crosslinked nanofibers mat as ceria nanoparticle carriers or a cohesive media. Despite the used deposition technique, ceria nanoparticles show that they are still active, as discussed in the next section.in situ in ceria NPs with concentrations of 0.25% and 0.75% are shown in A is a constant that depends on the effective masses of electrons and holes in ceria NPs, E is the absorbed photon energy, and gE is the allowed direct bandgap. E)2versus E, and the intersection of the extrapolation of the linear part of the (αE)2 curve with the E (x-axis) is equal to the allowed direct bandgap gE is the particular composition of ceria NPs.The absorbance curves of PVA NFs embedded in situ in PVA NFs. Studied concentrations of ceria NPs as examples were <1 wt %, 5 wt %, and 10 wt %. As shown in in situ are shown in Fluorescence intensity measurements were obtained for different concentrations of ceria NPs which were embedded in situ embedded ceria NPs at concentrations of 1 wt % and 5 wt % are studied, as are ceria NPs spin-coated on the mat of crosslinked PVA NFs as a post-addition step of the nanoparticles over the nanofibers, as shown in both in situ embedded ceria NPs and spin-coated ceria NPs on a mat of crosslinked PVA NFs, respectively. Regarding fluorescence intensity, as shown in in situ ceria NPs at 1 wt % and ceria NPs spin-coated on crosslinked PVA NFs have the same behavior as discussed in non-esterified NFs. That gives a promising conclusion that our electrospun nanocomposite can be optically fluorescent in addition to being hydrophobic. As discussed before, the esterification method is used as a chemical crosslinking method to convert the generated NFs from being a soluble mat to being a hydrophobic one that can resist dissolving in water. Here, the optical characterizations of crosslinked PVA NFs with in situ in PVA NFs that give a higher fluorescence intensity are less than 1 wt %.From in situ embedding technique than in the spin-coating one, due to the smaller obtained direct allowed bandgap and the higher value of the fluorescence intensity peak.As the optically effective materials are ceria NPs, the absorbance and bandgap curves in both All chemicals are used as received without further purification. Cerium (III) chloride heptahydrate (99.9%), Mowiol 10-98 Poly, Malic acid are purchased from Sigma-Aldrich . Methanol, ethanol and hydrochloric acid were purchased from an Egyptian local chemical agent  as commercial grade solutions.et al., but with some modifications [Undoped ceria nanoparticles are prepared using chemical precipitation technique similar to Chen ications . Cerium in situ to the PVA solution. The mixture is stirred for 30 min before it is entering the electrospinning process. Further, it would be shown another way to add ceria as post-synthesis treatment by spin-coating on the electrospun nanofibers.A concentration of 10 wt % PVA solution is prepared by mixing 10 g PVA pellets with distilled water of 90 mL. The solution is heated to 100 °C for 30 min then it was stirred overnight. This paper introduces different methods of producing PVA nanofibers with embedded ceria NPs. First, ceria NPs with different weight percentages of  are added The electrospinning setup consists of high voltage power supply , a syringe pump  which is used to regulate the pumping rate of polymer solution, a 5 mL plastic syringe with 18 gauge metallic needle to store the polymer solution, and a circular metallic collector of radius 10 cm covered with aluminum foil is used as a target. A schematic of electrospinning setup is shown in in situ with the PVA solution before being electrospun and esterified. Secondly, ceria nanoparticles were added as a post-synthesis step on the crosslinked electrospun PVA-only nanofibers through spin-coater, MTI-100 model , at a speed of 500 rpm.Vapor phase esterification process was done in oven on two subsequent steps similar to the procedure mentioned in Reference , but witin situ added or spin-coated, are optically characterized by measuring its optical absorbance, and fluorescence intensity curves. Optical absorbance in a wavelength within range from 300 to 700 nm was measured by using ultra violet-visible (UV-Vis) spectrophotometer of model (PG T92+) . From absorbance curves, corresponding band gap of the formed nanocomposite can be determined, as will be discussed in next section. Fluorescence intensity measurements have been detected by a hand-made fluorescence spectroscopy setup, as shown in PVA nanofibers with embedded ceria NPs, whether The mean synthesized nanoparticle size was observed by transmission electron microscopy  , with accelerating potential of 80 kV. Surface morphology and diameter size of electrospun nanofibers before and after esterification were investigated using scanning electron microscopy (FEI Quanta 200) . After sputter-coating with gold, the fiber size distribution of randomly selected SEM micrograph was measured using Image-J software. The formed nanocomposites have been characterized using FTIR spectroscopy (Varian 670-IR) .3+ ionization state have been added in situ to a PVA solution and then generated together as nanofibers via the electrospinning technique. In addition, to make the electrospun fiber hydrophobic mat, PVA nanofibers have been esterified using malic acid. In this case, ceria nanoparticles are added, whether in situ or on the esterified nanofibers as a post-synthesis treatment using spin-coating. In all synthesized nanofibers, the active ceria nanoparticles still keep their direct bandgap value around 3.5 V, which indicates that it is still optically active. All the synthesized nanofibers are shown to be visibly fluorescent under 430 nm optical excitation. The crosslinked nanofibers using esterification show a smaller mean diameter with greater number of beads compared to the non-esterified fibers. This new optical biodegradable nanofiber mat could be applicable in a wide variety of applications, including environmental sensors and cancer treatment.This paper presents the study of adding the property of optical fluorescence to electrospun PVA nanofibers through embedding ceria nanoparticles using different techniques. In this work, ceria nanoparticles with a greater Ce


All communities were dominated by cyanobacteria and among these 21 taxa were potentially endemic to any given desert location. Some others occurred in all but the most extreme hot and polar deserts suggesting they were relatively less well adapted to environmental stress. The t-RFLP and sequencing data revealed the two most abundant cyanobacterial taxa were Phormidium in Antarctic and Tibetan deserts and Chroococcidiopsis in hot and cold deserts. The Arctic tundra displayed a more heterogenous cyanobacterial assemblage and this was attributed to the maritime-influenced sampling location. The most abundant heterotrophic taxa were ubiquitous among samples and belonged to the Acidobacteria, Actinobacteria, Bacteroidetes, and Proteobacteria. Sequencing using nitrogenase gene-specific primers revealed all putative diazotrophs were Proteobacteria of the orders Burkholderiales, Rhizobiales, and Rhodospirillales. We envisage cyanobacterial carbon input to the system is accompanied by nitrogen fixation largely from non-cyanobacterial taxa. Overall the results indicate desert hypoliths worldwide are dominated by cyanobacteria and that growing season is a useful predictor of their abundance. Differences in cyanobacterial taxa encountered may reflect their adaptation to different moisture availability regimes in polar and non-polar deserts.Global patterns in diversity were estimated for cyanobacteria-dominated hypolithic communities that colonize ventral surfaces of quartz stones and are common in desert environments. A total of 64 hypolithic communities were recovered from deserts on every continent plus a tropical moisture sufficient location. Community diversity was estimated using a combined t-RFLP fingerprinting and high throughput sequencing approach. The t-RFLP analysis revealed desert communities were different from the single non-desert location. A striking pattern also emerged where Antarctic desert communities were clearly distinct from all other deserts. Some overlap in community similarity occurred for hot, cold and tundra deserts. A further observation was that the producer-consumer ratio displayed a significant negative correlation with growing season, such that shorter growing seasons supported communities with greater abundance of producers, and this pattern was independent of macroclimate. High-throughput sequencing of 16S rRNA and  Terrestrial ecosystems that experience prolonged moisture deficit are known as deserts or drylands and they comprise the largest terrestrial biome . They arA number of studies have reported biodiversity in hypolithic communities using microscopy and environmental sequencing approaches, and there is consensus from individual studies that they are dominated by cyanobacteria with a relatively cosmopolitan and less abundant heterotrophic assemblage in Africa , AntarctChroococcidiopsis identified that they displayed a high degree of endemism and could be divided phylogenetically into distinct hot and cold adapted clades . At each sampling location a minimum of three colonized stones were collected (Supplementary Table S1). The samples were obtained opportunistically in a crowd-sourced manner and so sample numbers for each desert region varied. Briefly, colonized quartz stones were retrieved by hand  and loose soil particles gently removed with a sterile (autoclaved) paintbrush. Samples were then stored in sterile Whirlpak bags (Nasco) at -20°C in the field and in transit, and subsequently stored frozen at -80°C in the laboratory until processed. Some subsequently failed to yield quality DNA and the final sample number analyzed was 64 hypoliths.Hypolithic communities were recovered as previously described  from quaSupplementary Table S1.Precipitation and temperature data were obtained from the freely available 10′ gridded global data sets provided by the Climate Research Unit (CRU) of the University of East Anglia . Mean ye2 area of colonized quartz stones using a sterile scalpel and forceps. Environmental DNA recovery was achieved separately for each sample by lysis in CTAB with lysozyme and RNAse, followed by phenol:chloroform extraction at 60°C  1, 1x PCR buffer provided by the manufacturer, 200 μM of each dNTP, and ddH2O. Amplification of 16S rRNA genes was achieved using primer pair 341F-CCTACGGGAGGCAGCAG and 907R-CCGTCAATTCMTTTGAGTTT . Fragment analysis was achieved by capillary electrophoresis , using a GeneScan ROX-labeled GS500 internal size standard from the manufacturer. The t-RFLP patterns and quality were analyzed using the freeware PeakScannerTM 3 and a data matrix comprising fragment size and abundance was generated. The software Perl and R  of Bray–Curtis similarities were generated from t-RFLP defined bacterial communities and groupings of communities in these ordinations were made with a 40% dissimilarity cutoff, using PRIMER 6 . The t-R4 according to manufacturer’s instructions. The libraries were quantified with Quant-iT PicoGreen dsDNA Assay Kit 2 using FLUOstar OPTIMA F fluorometer  5 and library quality was assessed with the FlashGel System . Emulsion-PCR was carried out with GS Junior Titanium emPCR Kit 6 according to the emPCR Amplification Method Manual – Lib-L, Single-Prep. The sequencing reaction was carried out with the GS Junior Titanium Sequencing Kit and GS Junior Titanium PicoTiterPlate Kit (454 Life Sciences Corp.) according to the manufacturer’s instructions. The sequencing run was conducted in 200 cycles. The nifH sequence libraries were generated using the primer set, nifH forward (5′ TGY GAY CCN AAR GCN GA3′) and nifH reverse (5′ADN GCC ATC ATY TCN CC3′) with barcodes in the forward primer. This followed the same methodology as the 16S rRNA pyrosequencing described above. All PCR reactions included positive and negative controls for 16S rRNA and nifH genes as appropriate.Samples for each statistically supported desert grouping from Bray–Curtis analysis (above) were selected based upon greatest number of shared OTUs from the t-RFLP analysis. These were further interrogated via barcoded pyrosequencing using the Roche GS Junior System . This resulted in high throughput sequencing of samples as follows: Taklimakan Desert, China; Tibetan Plateau, China; Devon Island, Canadian Arctic; McMurdo Dry Valleys, Antarctica. Amplification of 16S rRNA genes was achieved using primer pair 341F and 907R  with PCRde novo mechanism  revealed five distinct groupings at a 40% Bray–Curtis dissimilarity threshold . The tropical aquatic hypolithic communities were distinct from all desert samples. The Antarctic hypolithic communities were most distinct from all other communities worldwide. The three remaining desert groupings comprised a largely hot/cold deserts cluster, Tibetan Plateau cluster and Arctic/Sahara cluster. Some overlap between these three clusters occurred, for example hypolithic communities from Australia and California shared similarity across all three clusters, and Arctic and Saharan communities occurred in the same similarity cluster.Patterns in 16S rRNA gene defined diversity for 64 discreet hypolith communities . The most extreme deserts therefore likely supported communities with significantly greater photoautotrophic biomass as a relative percentage of the community than less extreme deserts with longer growing seasons. We identified a weak but significant inverse relationship between P/C ratio and mean annual precipitation , minimum winter temperature  and maximum summer temperature . A slightly more significant correlation was observed for growing season , a term extrapolated from combined macroclimate variables that describes the number of days per year when photosynthesis is possible.Resolution of t-RFLP sequence fragments with an extensive hypolithic sequence library  allowed SD = 42.25). The sequencing confirmed and validated the general trend in diversity and P/C observed in the t-RFLP study in that the Taklimakan Desert community was dominated by heterotrophic taxa with approximately 35% cyanobacteria, whilst in Tibetan and Arctic tundra deserts this figure rose to approximately 50%, and in the Antarctic sample cyanobacteria comprised over 70% of taxa . The dominant heterotrophic phyla in all samples were Acidobacteria, Actinobacteria, Bacteroidetes, Proteobacteria and Thermi, although the most abundant heterotrophic phylum varied between each sample . All four locations displayed a large number of unique OTUs with only eight OTUs shared across all locations and these were largely heterotrophic taxa . The geographically most proximal Taklimakan Desert and Tibetan Plateau samples displayed greatest shared similarity, closely followed by the Tibetan and Arctic samples . One sample (Arctic) had significantly more OTUs than all other samples but these were generally low abundance taxa comprising less than 1% of the overall community.In order to further understand the hypolithic biodiversity, we selected the most representative samples from each of the four statistically supported desert hypolith community clusters (based upon greatest number of shared OTUs) and subjected them to high throughput sequencing of the V3–V4 region of the 16S rRNA gene. Among the four samples, a total of 237,653 merged and filtered paired-end reads were generated for this study, with an average length of 344 bp . There were 21 taxa endemic to any given desert, whilst others occurred in all but the most extreme Taklimakan and Antarctic deserts. For example, OTU 2405 and 2434 were found only in geographically non-Polar deserts, OTUs 2114 and 2357 occurred in all but the extreme Antarctic desert, whilst OTU 2295 occurred in all locations except the Taklimakan Desert. A phylogenetic analysis of cyanobacterial OTUs  revealed that potentially endemic taxa spanned several cyanobacterial families. There was also a clear difference in the ratio of the two most abundant genera, Chroococcidiopsis and Phormidium (Table 1). In the Taklimakan desert virtually all cyanobacterial OTUs were Chroococcidiopsis, whilst in Antarctic samples they were almost exclusively Phormidium sp. Moreover the Antarctic desert was the only hypolithic community that did not support any Chroococcidiopsis OTUs.The sequence data indicated cyanobacterial taxa displayed a pronounced distribution pattern between the four desert samples (Table 1 and Supplementary Table S2). A second high throughput sequencing experiment targeting the nifH gene for nitrogenase was therefore conducted, although the number of filtered reads was low compared to the 16S rRNA study. Surprisingly no cyanobacterial nifH was recovered, instead all nitrogenase sequences affiliated with the Proteobacteria (Table 2 and Supplementary Table S3). This is despite recovery of 16S rRNA phylotypes indicating the diazotrophic family Nostocales occurred at low abundance in all locations except the hot desert.This pronounced difference in cyanobacterial distribution led us to ask whether aside from cyanobacterial photoautotrophy if nitrogen fixation, a function also traditionally associated with cyanobacteria in hypoliths and other communities, may be impacted by the biogeography of cyanobacterial assemblages. We screened the 16S rRNA sequences for taxa known to be diazotrophic, and revealed that Taklimakan, Tibetan and Arctic desert supported 14–18% putative nitrogen fixing taxa whilst in the Antarctic sample this dropped dramatically to approximately 5%  is known as a major adaptation to moisture stress. This confers advantage to the whole community thus they are a keystone species in hypolithic communities . The ecoWe introduced the application of a botanical concept, the growing season, a metric that reflects the number of days per year when a suite of macroclimate variables allow photosynthesis to occur . The P/CChroococcidiopsis in the Taklimakan Desert, and Phormidium in Tibetan and Antarctic deserts. The trend was pronounced between these three samples but the Arctic sample yielded a more diverse cyanobacterial assemblage dominated by poorly defined taxa largely from the Synechococcaceae. This may reflect the maritime location of this sample  since the genus Synechococcus has been identified in other maritime-associated desert hypoliths (Phormidium replaces Chroococcidiopsis as the dominant cyanobacteria as non-maritime deserts become colder and more extreme, such that in Antarctica hypolithic cyanobacteria are almost exclusively Phormidium sp. with no detectable Chroococcidiopsis. We rationalized the fast growing Phormidium is particularly adapted to Antarctic deserts where long periods of inactivity due to freezing are punctuated with short periods of favorable growth conditions where rapid colonization is an advantage, thus displaying elements of an R-selected strategy (Chroococcidiopsis taxa bear more resemblance to K-selected taxa that grow slowly but are specialized at exploiting highly xeric niches in hot and cold deserts due to copious EPS production and cellular desiccation tolerance mechanisms (Further consideration of cyanobacterial diversity revealed additional insight that may suggest a physiological basis for the observed trend in P/C ratio. Among the Cyanobacteria the notable observation was that cyanobacterial OTUs, whilst diverse, were largely dominated by two genera, ypoliths . It is istrategy . Converschanisms . The broChroococcidiopsis and Phormidium taxa to several other cyanobacteria across all families. Several taxa were encountered only in the two less extreme deserts whilst others were endemic to either extreme Taklimakan or Antarctic desert only. This supports the notion of stochastic demography for desert cyanobacteria observed in earlier studies (Acaryochloris was encountered in a single location only and this may indicate a relatively low-light microhabitat beneath quartz for these samples, as they have been recovered from low light habitats in marine and desert locations (Further potential patterns for cyanobacterial occurrence extended beyond the dominant  studies , and may studies , 2015. T studies . A pictu studies . This co studies  and so aocations . The chlocations . This isocations .nifH sequence library suggested a significant role for non-cyanobacterial diazotrophy by Alphaproteobacteria, and this has been inferred from 16S rRNA gene taxonomic data in earlier study of the polar environment (Nostoc and other diazotrophic cyanobacteria, the nifH library yielded known diazotrophic taxa only from within the Proteobacteria. The PCR primers used are known to amplify cyanobacterial nifH genes and so this requires further investigation to fully resolve the contributing taxa to nitrogen fixation.The ironment  as well ironment . Hypolitironment . This isironment . One surHypolithic microbial communities display patterns in diversity that appear to be driven largely by cyanobacterial adaptation to macroclimate and growing season. It seems reasonable to assume that diversity is determined by a complex interaction with multiple abiotic stressors and this could be a fruitful avenue for further research. The study also suggests that desert microbial communities may be far more diverse and regionally distinct than previously envisaged, and further work particularly with a focus on other domains of microbial life, may yield further insight . The stuSP, KW-R, and CM conceived the study. DL-B, ML, SP, and KW-R conducted fieldwork. DL-B, KL, SA, SL, and CL conducted laboratory experiments. DL-B, KL, SA, LG, TM, CM, JP, AdR-M, KW-R, DH, and SP analyzed data. DL-B, KL, SA, LG, TM, JP, AdR-M, KW-R, DH, and SP wrote the manuscript. All authors commented on and agreed the final 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.


Nonreinforced tensile repair of giant hiatal hernias is susceptible to recurrence, and the role of mesh graft implantation remains controversial. Creating a new and viable choice without the use of high-cost biological allografts is desirable. This study presents the application of dermis graft reinforcement, a cost-efficient, easily adaptable alternative, in graft reinforcement of giant hiatal hernia repairs.  A 62-year-old female patient with recurrent giant hiatal hernia (9 × 11 cm) and upside down stomach, immediately following the Belsey repair done in another department, was selected for the pilot procedure. The standard three-stitch nonabsorbable reconstruction of diaphragmatic crura was undertaken via laparoscopic approach. A 12 × 6 cm dermis autograft was harvested from the loose abdominal skin. “U” figure onlay reinforcement of diaphragm closure was secured with titanium staples. The procedure was completed with a standard Dor fundoplication. One- and seven-month follow-ups were conducted.  No short-term postoperative complications were observed. One-month follow-up showed normal anatomical location of abdominal viscera on computed tomography imaging. High-resolution manometry showed normal lower esophageal sphincter pressure. Preoperative abdominal complaints were resolved. Procedural costs were lower than the average cost following mesh graft reinforcement.  Dermis graft reinforcement is a cheap, easily adaptable procedure in the repair of giant hiatal hernias, even in the setting of laparoscopic reoperative procedure. As the number of surgical interventions required for gastroesophageal reflux disease (GERD) and/or hiatal hernias is increasing worldwide, surgeons encounter increasingly more complicated and inveterate cases with large hiatal defects. The number of publications per year related to hiatal repairs has almost doubled in the last decade .2 [Pearson et al., in their classification of hiatal hernias in 1983, defined giant hiatal defect as the herniation of 30% or more of the stomach into the thoracic cavity in the posterior mediastinum [2. After dubious chest X-ray findings, computed tomography (CAT) evaluation showed an “upside-down stomach” situated in the thorax with an organoaxial rotation, along with a 9 × 11 cm hiatal defect. Endoscopy showed LA-B erosive esophagitis and confirmed the nonanatomical location of the gastroesophageal junction. The patient underwent a Belsey Mark IV antirefluxplasty with hiatal reconstruction through left posterolateral thoracotomy at a thoracic surgery institute. On the 6th postoperative day, the patient developed severe dysphagia. Repeat CAT imaging confirmed paraesophageal reherniation of the antrum  reinforcement of the sutured hiatus using suture fixation, with results that encouraged further research . The newAutologous dermal grafts were one of the earliest biological matrices used in hernia repair in the early 20th century . While tDespite rapidly growing literature, there is still no gold standard for the type of graft to be used for reinforcement, in order to achieve the lowest rates of recurrence, complications, and procedural costs in giant hiatal hernias . We presThe only mentionable disadvantage is the scar formation at the harvesting site. This can be minimized with a proper suturing and wound care. If patient composition permits, the optimal harvesting site is the loose skin of the lower abdomen. However, some patients with giant hiatal hernia often experience difficulty of oral intake resulting in a decreased BMI; in these cases, consultation with a plastic surgeon is recommended.While comparing the additional burden of an abdominal skin scar to high-cost alternative grafts might be troublesome, the biological graft alternatives , 21 are Our department has utilized the autologous dermis graft in primal reconstruction of giant hiatal hernias in three more cases which reconfirm the excellent short-term results; data collection for prospective case series is in progress.We suggest onlay dermis graft reinforcement of the sutured diaphragmatic repair as a viable alternative in giant hiatal hernia repair. The method is eligible for further clinical evaluation. Further patient recruitment and long-term follow-up would be beneficial to determine the place of dermis graft reinforcement among the use of other biological matrices.


DNA methylation and the localization and post-translational modification of nucleosomes are interdependent factors that contribute to the generation of distinct phenotypes from genetically identical cells. With 112 whole-genome bisulfite sequencing datasets from the BLUEPRINT Epigenome Project, we analyzed the global development of DNA methylation patterns during lineage commitment and maturation of a range of immune system effector cells and the cancers that arise from them. We show clear trends in methylation patterns that are distinct in the innate and adaptive arms of the human immune system, both globally and in relation to consistently positioned nucleosomes. Most notable are a progressive loss of methylation in developing lymphocytes and the consistent occurrence of non-CG methylation in specific cell types. Cancer samples from the two lineages are further polarized, suggesting the involvement of distinct lineage-specific epigenetic mechanisms. We anticipate broad utility for this resource as a basis for further comparative epigenetic analyses. Different cell types stably express distinct phenotypes despite sharing an identical underlying genotype. Chemical modifications to DNA and associated histones allow genetically identical cells to exhibit radically different behavior and morphology by shaping gene expression programs and cellular responses to stimuli .Well-defined differentiation programs in the hematopoietic system provide an ideal model to investigate the mechanisms regulating cell identity. Lineage choice (myeloid or lymphoid) followed by further specialization and stable states of quiescence, activation, or long-term memory offer an established framework for studying epigenetic processes . PreviouDNMT3A and the demethylase TET2, are commonly mutated in cancer (DNA methylation is a ubiquitous epigenetic mark that is written directly onto DNA as the addition of a methyl group to a cytosine residue. Most DNA methylation occurs at cytosines followed by a guanine residue (CG dinucleotides), and the bulk of CGs genome-wide are methylated . Large-sn cancer . Many can cancer , and metn cancer  and of ln cancer , 2015. In cancer .Two key components of genomic organization that are reflected in, and affected by, methylation are the DNA-binding protein CTCF  and nuclNumerous experimental studies support the conclusion that nucleosomes protect occupied DNA from methylation, directing it preferentially to adjacent linker regions , and thaTo investigate the epigenetic mechanisms guiding cell identity and to provide a resource for the research community, the BLUEPRINT Epigenome Project has produced more than 200 whole-genome bisulfite sequencing (WGBS) DNA methylation maps, as well as genome-wide maps for six histone modifications, DNase-I hypersensitivity, and RNA expression for the large majority of these samples, covering the development of the major branches of the human immune system and several cancers that arise from them. Here, we integrated WGBS methylation maps for each of 112 samples from BLUEPRINT with CTCF-binding data from ENCODE and nucleosome occupancy derived from micrococcal nuclease digestion sequencing (MNase-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) data from BLUEPRINT samples and ENCODE cell lines.http://www. blueprint-epigenome.eu.We follow the development and activation of monocytes/macrophages and neutrophils in the myeloid lineage, and T and B lymphocytes in the lymphoid branch, and demonstrate consistent lineage-specific differences in the usage of DNA methylation globally and in relation to consistently positioned nucleosomes. We note progressive methylation shifts with differentiation and methylation trends in cancer samples that become more distinct between the two lineages. We also describe two distinct forms of apparent non-CG methylation: diffuse, globally high levels in naive T cells and uncommitted hematopoietic progenitor cells and dense exon-specific non-conversion in a few samples. All BLUEPRINT data are made available via −4; M1: p = 1 × 10−3; M2: p = 8 × 10−4), but the difference is small in comparison to the large differences among lymphocytes.Trends in global average CG methylation are markedly distinct between the lymphoid and myeloid lineages . FollowiIn both T and B lymphocytes, global methylation drops progressively with sequential stages of differentiation, resulting in the lowest genome-wide methylation levels in long-lived memory B cells and plasma cells, lymphoid cancers, and experimentally transformed B cells . The dynMethylation outside of a CG dinucleotide context  is prevalent in stem cells and neurons but was believed to be absent from other cell types . The non+ and three CD8+) and in uncommitted hematopoietic progenitor cells (UHPs) (−3) and is more similar to levels seen in stem cells. This widespread mCH disappears with subsequent stages of T cell development. Macrophage samples  also have elevated levels of mCH relative to most committed cell types but do not reach the levels seen in progenitors and naive T cells. Leukemias derived from the myeloid lineage (acute promyelocytic leukemia [APL] and acute myeloid leukemia [AML]) appear to consistently gain considerable mCH . In lymphoid-derived neoplasms , mCH appears more varied  . The frae varied . Cancer Diffuse mCH is found overwhelmingly in a CA context (>90%), with CAC accounting for 72% of methylated non-CG cytosines in naive T cells . CAC is −6). Because LADs cover 1/3 of the genome and are gene poor, mCH exclusion from these regions may contribute to the observed enrichment in genes.In all hematopoietic samples where it occurs at appreciable levels, non-CG methylation is enriched in genes (exons and/or introns), to varying degrees. At the scale of whole chromosomes, the most distinctive feature of the high levels of mCH is their exclusion from lamina-associated domains (LADs) , occupied only in specific cell types, or unoccupied, as in In each sample where it occurs, mCH follows a similar nucleosome-influenced spatial enrichment pattern as CG methylation near cCTCF, but the bias for mCH toward linker regions is even more pronounced than for CG methylation, with almost complete exclusion from nucleosome-occupied DNA . AggregaIn line with our methylation results, the oscillation of the nucleosome positioning signal at cell-type-specific CTCF sites is much flatter than the strong alternating enrichment and depletion characteristic of the cCTCF sites. We observe regular positioning surrounding cCTCF sites using nucleosome occupancy data derived from MNase digestion in either a lymphoid or myeloid cell line; we further confirm this observation using nucleosome positions derived from the integration of ChIP-seq data for six histone modification marks  obtainedVisual inspection of In both B and T lymphocytes, the oscillatory methylation pattern at cCTCF sites becomes more pronounced with successive stages of development while global average methylation levels decline . DiffereIn contrast to the methylation trends seen in developing lymphocytes, the oscillating methylation pattern near cCTCF in the myeloid lineage is less varied and shows no consistent trend .Neoplastic transformation results in increased lineage-specific differences in linker methylation bias . CancersDNA methylation has a direct role in regulating cell phenotype, for example, by enforcing CD4 repression that defines the difference between cytotoxic and helper T cell subsets . We and During hematopoietic development, DNA methylation patterns diverge. Methylation bias toward nucleosome-free DNA increases progressively with T and B lymphocyte development while global methylation levels steadily decline. In contrast, CG methylation appears quite stable during differentiation and activation of both neutrophils and macrophages in the myeloid lineage. Declining global methylation levels in developing lymphocytes likely reflect the emergence of partially methylated domains, which to our knowledge have not been reported in normal cells of the myeloid lineage.Methylation measurements in leukemias and lymphomas from both lineages are heterogeneous, consistent with the genetic heterogeneity typically observed in cancer samples. The methylation profiles of malignant cells are likely to vary substantially from cell to cell and among samples from different individuals. However, we see general trends here as well. As a group, lymphoid-derived neoplasms continue to lose CG methylation, whereas myeloid cancers have significantly elevated levels of mCH. This observation has potential clinical relevance, as demethylating agents used as chemotherapeutics  could haThe diffuse non-CG methylation we observe is strongly biased toward linker DNA, strongly depleted from LADs, and occurs specifically in all naive T cell samples and the UHPs. Although the spatial enrichment patterns reflecting nucleosome occupancy are similar for mCG and mCH, the occurrence of high levels of diffuse mCH only in specific cell types suggests some degree of independence in the processes governing methylation in CG and non-CG contexts. The cell-type-specific occurrence and association with structural features of the genome further support the interpretation that mCH is a biological phenomenon and not merely a technical artifact.We showed that mCH levels across samples and cell types are not dependent upon bisulfite conversion rate, given a passably effective bisulfite treatment. Therefore, we suggest that bisulfite conversion efficiency for WGBS studies should be reported based on proper controls using known fully methylated and fully unmethylated DNA and not as the fraction of non-converted non-CG cytosines, as commonly done.We note that many of the same cell types high in mCH, such as naive T cells, stem cells, and neurons, also have higher reported levels of hydroxymethylation . As thisThe vast majority of mCH occurs as CA dinucleotides. Deamination of unmethylated cytosine results in uracil, which is efficiently recognized and repaired, but deamination of methylated cytosine (mC) produces thymine. In a mCG dinucleotide context, deamination on one strand creates a T:G mismatch that may be repaired either to the original C:G pair or to a new T:A pair, yielding a mCA dinucleotide on the strand opposite the deamination event. CA dinucleotides resulting from this process  may remain susceptible to methylation under certain conditions, such as high local concentration or polymerization of DNA methyltransferases . This maThe biochemical basis of non-CG methylation in human DNA is unclear. In mouse embryonic stem cell lines, depletion of either DNMT3A or DNMT3B decreases mCH, whereas depletion of DNMT3L, which does not have a functioning methyltransferase domain, increases mCH levels . In contThe molecular mechanisms of CG methylation have been described using structural and functional studies . AlthougLimited DNA accessibility due to nucleosome occupancy or association with the nuclear lamina provides a simple explanation of the methylation patterns we observe, both CG and non-CG. Alternatively, DNA methylation may occur in conjunction with nucleosome translocation as part of an active remodeling process that leaves methylated DNA in its wake, as already demonstrated in vitro . Local DDNMT1 and DNMT3 and a chromomethylase . Five samples were excluded based on conversion rates <0.997, supported by visual inspection of CG and non-CG methylation plots. The over-conversion rates for all samples based on methylated phage T7 DNA were ∼5%.Methylated non-CGs were defined as those cytosines with at least two unconverted reads and a methylation probability greater than two SDs, where the probability of the following base being a guanine was at least two orders of magnitude smaller than the most probable base. The fraction of mCH for each sample is calculated as the number of methylated non-CG cytosines divided by the total number of non-CG cytosines with six or more reads informative for methylation status.Spikes are called in mCH data where the mean methylation value in a sliding window of ten non-CG cytosines exceeds 20%. Only non-CGs with six or more reads informative for methylation are considered.Additional WGBS data for stem cell lines H1 and H9 were generated by the Roadmap Epigenome Project . WGBS daPairwise sample comparisons were made to determine which sites underwent significant changes in methylation level at each step of B lymphocyte development. All positions that passed quality filtering and were called as homozygous CGs in both samples were evaluated. Significance in the difference between samples in the ratio of converted to unconverted reads at each CG was assessed using Fisher’s exact test if any expected values of the contingency table were less than ten. Otherwise, a chi-square test was used. Results were adjusted for multiple comparisons using the false discovery rate method .To facilitate high-throughput comparison of many samples, an automated procedure was developed to measure the amplitude of the oscillating methylation signal. To measure amplitude, we must first determine the distance from the CTCF-binding motif of the first peak and the first valley in the methylation signal. Both the period of oscillation and the average methylation levels differ among samples and cell types, so measuring amplitude assuming a fixed period or by using the difference between the maximum and minimum methylation values within a specific distance are unlikely to give good results. Our signal is further complicated by noise and periodic components at multiple scales. Wavelet-based transformation is commonly used in digital signal processing to smooth noisy data and to identify and localize periodic components, making it ideal for this purpose.A multi-resolution analysis  using thAlthough we refer to oscillation amplitude in terms of nucleosome influence on methylation, we note that it is determined based only on the methylation signal and does not consider explicit information about nucleosome position.Nucleosome positions for ENCODE cell lines GM12878 (lymphoblastoid) and K562 (myeloid) were inferred from MNase sequencing reads, normalized to input read levels described in Whole-genome DNA methylation and nucleosome positioning data were aligned to CTCF-binding motifs and plotted for visual inspection. To preserve binding orientation information for CTCF, data surrounding motifs that occurred on the minus strand were reversed. Non-CG methylation levels were computed separately for the strand containing the CTCF-binding motif and the opposite strand. Methylation levels for the opposite strand are plotted as negative values to facilitate visual analysis.Occupied CTCF-binding sites were identified by http://www.blueprint-epigenome.eu.Data generated by the BLUEPRINT project are available through several channels, including genome browsers, Biomart, and directly through the BLUEPRINT portal. Links to data sources are available at the BLUEPRINT website: http://dx.doi.org/10.1016/j.celrep.2016.10.054.Supplemental Information includes six figures and one table and can be found with this article online at Supplemental information


Cyclic dinucleotides that activate STimulator of INterferon Genes (STING) have been shown to cause rapid regression of a range of advanced tumors. We aimed to investigate STING ligands as a novel treatment for papilloma. We tested therapies in a spontaneous mouse model of papilloma of the face and anogenital region that histologically resembles human HPV-associated papilloma. We demonstrate that STING ligands cause rapid regression of papilloma, associated with T cell infiltration, and are significantly more effective than Imiquimod, a current immunotherapy for papilloma. In humans, we show that STING is expressed in the basal layer of normal skin and lost during keratinocyte differentiation. We found STING was expressed in all HPV-associated cervical and anal dysplasia and was strongly expressed in the cancer cells of HPV+ HNSCC but not in HPV-unrelated HNSCC. We found no strong association between STING expression and progressive disease in non-HPV oral dysplasia and oral pre-malignancies that are not HPV-related. These data demonstrate that STING is expressed in basal cells of the skin and is retained in HPV+ pre-malignancies and advanced cancers, but not in HPV-unrelated HNSCC. However, using a murine HNSCC model that does not express STING, we demonstrate that STING ligands are an effective therapy regardless of expression of STING by the cancer cells.Human papilloma virus positive (HPV As our ability to detect cancerous cells in patients improves, there is an opportunity to treat early tumors before they become invasive and metastatic. Most cancers are known to exhibit a premalignant state, where an expanded population of abnormal cells disrupts normal tissue organization. This premalignant state can exist for years and may remain, resolve, or progress to invasive cancer. Cancer screening programs—for example those based around the Papanicolaou (Pap) smear for cervical carcinoma—aim to identify these abnormal cells for intervention before further malignant transformation. Vaccination against HPV can prevent HPV infection and thus, in principle, prevent HPV-associated malignancies. However, once tumors develop, vaccination against the virus has limited potential impact on progression, perhaps because prophylactic vaccination is directed to neutralizing antibody response to free virions rather than effector T cell control of infected cells . CurrentKras(G12D) and Tp53(R172H), leading to progressive carcinogenesis from pancreatic intraepithelial neoplasia [In the transgenic pancreatic ductal adenocarcinoma mouse model, Cre expression under a pancreas-specific promoter activates expression of the mutant tumor-driving genes eoplasia . In addieoplasia , 5. The -associated premalignancies and advanced cancers, but not in HPV- HNSCC derived from non-basal cells. Since treatment with STING ligands causes rapid regression of spontaneous murine papilloma this may represent an advance in the treatment of virus-associated and premalignant diseases. However, STING expression by malignant cells is not essential for treatment of advanced cancers.Recently, we and others have demonstrated that cyclic dinucleotide (CDN) ligand activation of the STimulator of INterferon Genes (STING) pathway strongly induces type I IFN and TNFα, resulting in rapid regression of a range of advanced tumors , 7. We tAll animal protocols were approved by the Earle A. Chiles Research Institute IACUC . De-identified human tissue sections were obtained under IRB# 12–075 approved by the Providence Portland Medical Center IRB.+/- , Kras(G12D)+/- , and Trp53(R172H)+/-  mice were crossed to generate Pdx-Cre+/- Kras(G12D)+/- Trp53(R172H)+/-that generate pancreatic tumors [Pdx-CreTo assess the cytokine response of papilloma to STING ligand, papilloma were removed from mice, dissected under sterile conditions into 1-2mm fragments and these were placed in complete media in the presence or absence of 25μg/ml CDN. The papilloma explants were incubated overnight at 37°C then supernatants were collected for multiplex cytokine analysis. Cytokine levels in the supernatants were detected murine multiplex bead assays  and read on a Luminex 100 array reader. Cytokine concentrations for replicates of each sample were calculated according to a standard curve.Archived tissue blocks were obtained and 5μm sections were cut and mounted for analysis. Tissue sections were boiled in EDTA buffer for antigen retrieval. Sections were first stained with rabbit anti-STING  and primary antibody binding was detected with HRP conjugated secondary antibodies followed by DAB development and counterstaining. Images were acquired using a Leica SCN400 whole slide scanner. For immunofluorescence staining, sections were stained with anti-CD3  and primary antibody binding was visualized with AlexaFluor 568 conjugated secondary antibodies  and mounted with DAPI  to stain nuclear material. Images were acquired using a Zeiss Axio observer Z1 with attached Nuance Multispectral Image camera and software . All images displayed in the manuscript are representative of the entire section and their respective experimental cohort.Representative blocks of archived tissue from 5 patients for each histology were obtained and sectioned. In order to represent distinct types of oral dysplasia, we identified examples of benign dysplasia, inflammatory conditions including lichen planus and candida ulcer, as well as examples of mild, moderate and severe dysplasia. To represent HPV-asssociated dysplasia and premalignancy we identified examples of benign dysplasia, chondyloma, anal intraepithelial neoplasia grade 3 (AIN3), and cervical intraepithelial neoplasia grade 3 (CIN3). Tissues were sectioned and stained for STING as above. The degree of STING staining was scored by the reviewing pathologist.+ if they scored as positive for p16 and originated in the oropharynx. Samples that were negative for p16 and/or originated outside the oropharynx were scored as HPV-. Arrayed tissues were sectioned and stained for STING as above. The STING staining score was determined by automated image analysis of tumors, grading the number of STING positive cancer cells and their staining intensity to generate an expression score.A panel of paraffin embedded tissue blocks was used to generate an array of tumors of mixed HNSCC origins using a Perkin Elmer tissue microarrayer. Patient tumors were classified as HPVk).SCCVII were transfected with a plasmid vector expressing murine STING  and grown under antibiotic selection to generate stable clones expressing STING. To confirm expression, cells were lysed in RIPA buffer and denatured in SDS loading buffer containing β2-mercaptoethanol, electrophoresed on 10% SDS-PAGE gels and transferred to nitrocellulose. Blocked blots were probed overnight at 4°C with primary antibodies followed by HRP-conjugated secondary antibodies. Binding was detected using a Pierce SuperSignal Pico Chemiluminescent Substrate  and exposure to film. To confirm response to STING ligands, cells were left untreated, treated with 25μg CDN or with 10ng/ml IFNγ. 24hr later cells were analyzed by flow cytometry for expression of MHCI . Individual data sets were compared using Student’s t-test and analysis across multiple groups was performed using ANOVA with individual groups assessed using Tukey’s comparison.+/- Kras(G12D)+/- Trp53(R172H)+/- mice were observed to develop papilloma at variable penetrance, as has been previously reported [(G12D) genotypes suggesting a genetic origin [- animals nor PDX-Cre+ Trp53(R172H)+/- mice that lacked Kras(G12D). Mice were unperturbed by the papilloma, and though individual papilloma could attain large size, we found no evidence of progression to invasive carcinoma, although tandem progression of pancreatic adenocarcinoma in these mice precluded long-term follow up of the papilloma, per se. Histological analysis of the papilloma demonstrated significant thickening of the skin with formation of classical keratinizing papilloma as reported [Pdx-Crereported , 5. We fFig 1Ai). These small CDN-injected papilloma rapidly reverted to skin with normal appearance following treatment . The site exhibited slight reddening, and histological analysis demonstrated an inflammatory infiltrate in the subcutaneous space . Interestingly, injection of STING ligand into histologically normal skin at distant sites or in normal mice had no observable effect (not shown). Thus, CDN injection appears non-toxic at these doses and causes rapid, site-specific regression of experimental papilloma.To determine whether the papilloma could be treated using STING ligands, mice bearing papilloma were randomly assigned to treatment with a STING activating cyclic dinucleotide (CDN), or PBS vehicle control. In an example of a mouse with two small papillomas on the face, one was treated by direct injection of CDN, and the other given vehicle control . Mice were randomized to receive CDN, conventional treatment with the TLR7 ligand Imiquimod or PBS vehicle. While Imiquimod did not significantly affect the size of the papilloma, CDN treatment resulted in significantly reduced papilloma size  (p<0.001 days 4–15). The CDN-treated papilloma showed evidence of blackening , which was suggestive of the hemorrhagic necrosis we have previously observed in STING ligand treatment of advanced cancers [We then tested treatment of larger, 3-15mm papilloma with STING ligands. A single injection of  cancers . Treatme+ T cells. Few to no T cells were detected in PBS or Imiquimod-treated papilloma , but 24 hours following CDN treatment CD3+ T cells were found associated with the epidermis in the treatment site . In examples where mice exhibited more than one papilloma, CDN treatment resulted in T cell infiltration in the treated papilloma with poor infiltration to untreated sites . This is consistent with the observed site-specific response to CDN injection . T cells remained enriched in the epidermis of the treatment site 14 days following treatment . Control of papilloma by CDN treatment occurred regardless of the presence of CD8+ T cells , consistent with an innate rather than an adaptive immune control of disease. To measure innate cytokines that were released following treatment with STING ligands, explants of papilloma were treated with STING ligands and cytokine secretion was measured by multiplex bead assay. Fig 2D)Treatment with STING ligands resulted in a significant secretion of IFNβ, and a trend towards increased TNFα secretion, but this did not reach statistical significance . However, endothelial cells and infiltrating immune cells underlying the papilloma strongly express STING , and these cells are also the likely target for STING ligands [To investigate whether the response involved adaptive immunity, papilloma were stained for infiltrating CD3ss STING , and STIss STING ). Histol ligands , 7.Fig 3. Immunohistology demonstrated strong expression of STING in cells of the tonsil , in particular, strong staining was observed in the high endothelial venules , in cells within the light zone of the germinal center that are consistent with follicular dendritic cells , and in cells within the T cell zone that are consistent with interdigitating cells . STING expression was readily detectible in immune cells underlying normal tonsillar epithelia, but was particularly high in select tonsillar crypts , suggestive of ongoing local responses to bacterial or viral exposure. In normal tongue, STING expression was restricted to the basal cell layer and lost upon differentiation to keratinocyte layers . In some areas of oral candidiasis associated with benign thickening, STING expression was enhanced . In mild or severe dysplasia of the oral tongue, STING expression was mixed but did not increase with the degree of dysplasia , and occasionally STING expression was not detected in the squamous cells of severe oral dysplasia . However, in each case, expression of STING was readily detectable in endothelial cells and in immune cells in the submucosal layer underlying the squamous layer. These data show that STING is expressed by basal cells in the oral mucosa, which are a key target for HPV infection, but there is no significant association with STING expression and the progression of dysplasia in the tongue or oral cavity.To understand whether this expression pattern is shared in patients, we examined STING expression in normal human tonsil and in a panel of human benign and premalignant oral dysplasia. Representative examples are shown in + neoplasia are rarely observed in either site. To evaluate STING expression in HPV-associated premalignancies, we performed immunohistology of high grade cervical intraepithelial neoplasia (CIN3), high grade anal intraepithelial neoplasia (AIN3), and condyloma, as well as benign hyperplasia and tissues from normal uterus as a control. The pattern of normal tissue STING expression was very similar to that seen in the tonsil and tongue, with predominant expression in the basal cells and underlying immune cells, and loss upon differentiation into the keratinocyte layer . STING was also expressed in all HPV-associated dysplasia, with a trend towards increased expression of STING in CIN3 . In addition, in all cases, STING was expressed in endothelial structures and immune cells underlying the squamous epithelium. These data demonstrate that STING is expressed in basal cells and expression is maintained or increased in these cells through HPV+ dysplastic progression.While HPV is known to result in HPV-associated cancers in the palatine and lingual tonsils, premalignant HPV+ and HPV- cancers, we stained a tissue array from a panel of HNSCC from various anatomic sites. The tumors were classified as HPV+ if they scored as positive for p16 by immunohistochemistry and originated in the oropharynx. Samples that were negative for p16 and/or originated outside the oropharynx were scored as HPV-. In all cases, STING was detectable in immune cells in the vicinity of the tumor. However, HPV+ HNSCC exhibited high levels of STING expression in the cancer cells , while HPV- HNSCC cancer cells exhibited low or absent STING expression . This expression was quantified using image analysis software according to the proportion of cancer cells with high STING staining in the cytoplasm, confirming that HPV+ HNSCC exhibited significantly higher STING expression in cancer cells than HPV- HNSCC . These data suggest that HPV+ cancer cells preserve STING expression through malignant progression from their basal cell origin, in contrast to HPV- HNSCC, which is generally composed of keratinizing, non-basaloid epithelium that poorly express STING. This is consistent with the loss of STING expression during normal squamous differentiation.To examine STING expression in advanced HPV- HNSCC; however, host rather than cancer cell expression of STING has been shown to be critical for therapies targeting STING [Fig 6Ai) in contrast to our prior work with the Panc02 pancreatic adenocarcinoma cell line that expresses STING and can directly respond to STING ligands in vitro [Fig 6Aii). Stable expression of STING in these cells restores responses to direct treatment with STING ligands , confirming that unmodified SCCVII cells cannot directly respond to STING ligands. Despite increases in MHC following treatment with CDN, secretion of type I IFN was below the detection level of our assay, suggesting that the engineered cancer cells are weakly responsive to in vitro stimulation. To evaluate whether STING unresponsive SCCVII tumors could respond to treatment with STING ligands in vivo, unmodified parental SCCVII tumors were established in immunocompetent C3H mice and once established, treated with 2 daily injections of PBS alone or 25μg CDN in PBS. CDN treatment resulted in a rapid, though transient tumor regression, which significantly enhanced survival of these mice . These data demonstrate that STING ligands are effective where cancer cells lack STING expression.These data would appear to suggest that therapies targeting STING would be ineffective in HPVng STING , 10. To ng STING . We idenin vitro and in vivo, and can result in dramatic regression of tumors in mice. We demonstrate in a mouse papilloma model that the STING ligands are superior to Imiquimod for regression of papilloma, yet is non-toxic to normal skin. We demonstrate that STING is expressed in the basal layer of normal tonsil and in tonsillar crypts, which are a presumed site of HPV infection. STING expression is preserved in HPV-associated premalignancies and in HPV+ HNSCC, but lacks association with non-HPV oral dysplasia and HNSCC. Nevertheless, via effects on host cells STING ligands can treat tumors derived from cancer cells that lack STING expression.Despite anecdotal efficacy and good results in single arm studies, Imiquimod and type I IFN have not demonstrated efficacy in randomized clinical studies of HPV-associated premalignant disease. There remains a need for alternative agents to treat HPV-associated disease and prevent progression into invasive cancer in high-risk groups. Ligands targeting the STING pathway have been shown to activate potent inflammatory responses +/- Kras(G12D)+/- Trp53(R172H)+/- model of papilloma, in particular the ongoing progression of tandem pancreatic tumors. However, the genetic development of papilloma in these mice is similar to other squamous cell carcinoma murine models. A number of systems are based on the K14 promoter to ensure activation of oncogenes in basal keratinocytes. Mice with K14 driven expression of Cre bred with the same Kras(G12D)fl/fl floxed mice used in our study, develop variably situated benign papilloma and additional cross with the same Trp53(R172H)fl/fl mice used in our study resulted in variably situated invasive carcinoma [+/- Kras(G12D)+/- Trp53(R172H)+/- mice have the potential to become invasive carcinoma were it not for the dominant progression of the lethal pancreatic tumors. Mutation of Ras family members and mutation or loss of heterozygosity of Trp53 are commonly detected in models of mutagen-driven squamous cell carcinoma [in vivo and measure their ability to prevent the development of invasive carcinomas in immune competent animals. However, in view of our data that STING is expressed in the basal cell layer of the papilloma, but that this is lost on differentiation to keratinocyte layers, this may explain why HPV positive tumors express STING whereas HPV negative tumors do not. HPV is known to infect the basal layer of stratified epithelium whereas non-HPV squamous cell carcinoma tends to originate from the more differentiated keratinizing layers of the epithelium. We propose that STING expression is linked to the basaloid squamous cell origin of HPV+ cancer, and is not a result of HPV infection or due to expression of HPV-associated oncogenes. Therefore, the presence or absence of STING expression in these alternative models will be determined by the cell origin of oncogenesis rather than the presence of HPV genes.We acknowledge limitations inherent to the Pdx-Cre+/- KrasGD+/- Trp5arcinoma , and thearcinoma . K14-driarcinoma , the ratarcinoma . Similar/- Trp53R2H+/- modarcinoma . Models - HNSCC, this does not necessarily preclude ligands targeting STING as a therapeutic approach in HPV- HNSCC. We have previously shown that STING expression in the host and not the cancer cell is critical for responses to STING ligands and radiation therapy [- HNSCC model, STING expression by the cancer cell is not essential for the response to STING ligands. We see similar responses to STING ligands in tumors where the cancer cells express STING as in those where the cancer cells do not express STING [Recent data suggests that cancer cell expression of STING is critical for the full effect of radiation therapy (19). While this is not clear evidence for the therapeutic responsiveness of HPV positive head and neck cancer, it is one possible explanation for the difference in response that is seen clinically. In the papilloma model it is probable that the oncogene-driven benign papilloma lack the mutated neoantigenic targets that would be necessary for an effective T cell-mediated adaptive immune response [per se, and is consistent our data showing induction of type I IFN in the papilloma. We previously demonstrated that early control of invasive carcinoma treated with STING ligand was dependent on innate cytokines and was preserved in CD8-depleted mice as well as Rag-/- mice lacking T cells [+ and HPV- tumors. We and others have shown that myeloid cells respond strongly to STING ligands [in vivo. Thus, treatment with STING ligands may depend on host cells producing TNFα and type I IFN, and these cytokines then act as effectors. However, we cannot at present exclude direct recognition of STING ligands by STING expressed in abnormal basal cells as the cause of papilloma regression in our model. Further studies are necessary to evaluate the mechanisms of papilloma control, the relative role of innate cytokines, adaptive immunity and the relative response rates in STING+ and STING- cancers.While we show that STING is not frequently expressed in HPV therapy  and othe therapy . As we dss STING . In thesresponse . Adaptiv T cells . However T cells . Therefo therapy . While t T cells , 21. If  T cells . In the  ligands , 7, and While our major goals in developing this therapy are preventing progression of premalignant disease to lethal invasive cancer and treatment of invasive cancer, non-progressing benign HPV-associated papilloma remain a significant public health concern. The non-ablative rapid regression of papilloma following application of STING ligands could be highly applicable to condyloma, verrucae, or warts. Moreover, since this therapy was effective without onboard viral-associated genes, this therapy could be applicable to intraductal papilloma and choroid plexus papilloma that are of unknown cause. We propose that STING represents a novel innate immune target for treatment of benign dysplasia and premalignancy involving basal cells, in addition to its emerging role as a therapy for invasive carcinomas.