BRCA) mutation with reduced ovarian reserve, the data is yet inconsistent. Our aim was to investigate biomarkers of ovarian aging in a cohort of young healthy carriers of the BRCA mutation. We hypothesized that the role played by BRCA genes in aging pathways is not exclusive to the ovary.Though former evidence implies a correlation of breast cancer susceptibility gene , fibroblast growth factor-23 (FGF-23), Klotho and IL-1 were measured by enzyme-linked immunosorbent assay (ELISA). Ovarian AMH and protein kinase B (AKT) mRNA from BRCA carriers who underwent prophylactic oophorectomy and from age-matched, healthy, non-carriers who underwent partial oophorectomy due to benign conditions were analyzed by qPCR.Healthy female BRCA carriers were enrolled into the study and matched to control non-carriers . Serum AMH level was significantly lower in BRCA female carriers than in both non-carrier controls and age-matched nomograms. The levels of ovarian AMH and AKT mRNA were significantly lower in carriers than in controls. The systemic aging cytokines FGF-23, klotho and IL-1 displayed a differential expression in carriers of both genders. FGF-23 level was higher in carriers (P=0.06).Thirty-three female (median age 35y) and 20 male (44y) BRCA mutation, accelerated ovarian aging and systemic aging-related pathophysiology.Our results suggest a link between  BRCA1) and 2 (BRCA2), predispose their carriers to a higher lifetime-risk of breast and ovarian cancers [BRCA1/2 mutations, include male breast cancer, prostate cancer, pancreatic cancer, and melanoma [BRCA1 or BRCA2 display higher levels of cellular senescence and apoptosis and are embryonically lethal [BRCA1+/− p53+/− mice displayed multiple premature aging phenotypes, such as osteoporosis, atrophy, kyphosis, decreased body weight, and increased tumor incidence [Germline mutations in the two known breast cancer susceptibility genes; breast cancer susceptibility gene 1  and 20 male carriers (44y) were enrolled and matched to 15 non-carrier females (34y) and 16 non-carrier males (43y) as depicted in Table BRCA carriers than in female non-carrier controls and continue to decrease with age . Interestingly, AMH level was similar in BRCA carriers and non-carriers . Moreover, the levels of AKT and AMH mRNAs as indicators of cell survival and proliferation [BRCA carriers than in non-carriers .Thirty-three female carriers ; P>0.05.s Figure ; P<0.05;feration  and of os Figure ; P<0.05.BRCA carriers in order to examine possible involvement of BRCA in systemic aging. FGF-23 is a key molecule regulating mineral homeostasis and vascular calcification and is its concentration in serum is correlated with aging [BRCA carriers of both genders than in the control group, though nearly significant (P=0.06 CI), possibly because of high variability. Because there are no established cut-off values for the level of serum FGF-23 we normalized the level of FGF in the serum to the median in our cohort. The distribution range of FGF-23 values among carriers was significantly different in BRCA-carriers and non-carriers. Only one non-carrier had an FGF-23 level higher than 75th percentile, whereas 8 BRCA-carriers depicted outlier values , compared to age-matched non-carriers. Klotho levels in both genders were lower in the serum of BRCA carriers than in non-carriers, though they did not reach statistical significance. The levels of IL-1A in the serum depicted a reciprocal pattern: higher in BRCA carriers, though due to high variability it also did not reach statistical significance. Limited by small sample size, the patterns yielded in our study were very obvious, yet not clearly significant, hampered by high variability. Aging is also characterized by increased cardiovascular risk, due to endothelial dysfunction via increased generation of reactive oxygen species (ROS), thus elevating oxidative stress that in turn promotes DNA damage and endothelial cell apoptosis [BRCA mutation carriers had increased risk of chemotherapy-induced cardiotoxicity in both mouse and human [BRCA as a gatekeeper of inflammation-induced endothelial cell function and a target to limit atherosclerosis [BRCA1 is expressed in endothelial cells in mice and overexpression of BRCA1 strongly attenuated the production of ROS and AKT. Moreover, a Canadian study estimated that in the absence of melanoma, breast, ovarian and pancreatic cancers, the life expectancy of female and male BRCA carriers was 6.8 and 3.7 years lower than that of non-carriers, respectively [BRCA1/2 mutations and increased non-cancer mortality, complementing our data.The link between ovarian function and aging has been well established. Menopause transition has been correlated with aging and increased risk of future cardiovascular morbidity –33. AMH,carriers . We presic aging , 27–28 opoptosis . It had nd human , 42. Theclerosis –46. BRCAectively . An overBRCA mutation, accelerated gonadal aging and systemic aging-related pathophysiology. Because DNA damage and repair plays an essential role in carcinogenesis, it also represents a common contributor for aging-related conditions characterized by increased cellular and oxidative stress manifested by DNA damage. Thus, BRCA carriers may be predisposed to enhanced aging, potential endothelial dysfunction and related vascular morbidity. Future studies should elucidate this hypothesis and appraise the prevalence of non-cancer morbidities in BRCA carriers, as these data may carry a significant implication for preventive and early detection measures for this population.In conclusion, our results lend credence to a newly paradigm, that suggests a potential link between BRCA carriers; 33 females, 40 years or younger, before prophylactic oophorectomy (PO) and 20 males, 50 years or younger. Control cohorts were consisted of age-matched healthy non-carriers of each gender . The protocol was approved by Rabin Medical Center (RMC) institutional review board (RMC-6103) and all patients signed a relevant informed consent form. Blood samples were drawn from both genders for hormones and cytokines measurements. Ovarian tissue obtained from BRCA carriers who underwent PO, and from age-matched healthy non-carriers, who underwent partial oophorectomy due to benign conditions, were processed for histological and gene expression analyses.Study participants were healthy Blood samples were centrifuged  and serum was stored in aliquots at −20°C until measurements. Anti-Mullerian hormone , fibroblast growth factor-23 , interleukin 1 alpha  and Klotho  were measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions. Calibrators for a standard-curve as well as low and high controls were added in duplicates to each ELISA plate. Samples were measured by the SpectraMax 190 microplate-reader .2 of ovary was recorded and presented as percentage of follicular density in control group. The total volume of the ovaries was not assessed.Ovaries were fixed in 4%; paraformaldehyde , embedded in paraffin, sectioned (8 μm) and mounted on Superfrost/Plus slides . Several transverse sections from each ovary were stained with hematoxylin and eosin (H&E). Bright-Purification of total RNA from formalin fixed, paraffin-embedded microdissected ovarian tissue was made by RNeasy-FFPE kit according to the manufacturer’s instructions . Ovarian mRNA was quantified ; first-sP < 0.05. A correlated one-way ANOVA statistical analysis showed similar results. Data of detected or undetected serum FGF-23 levels was also evaluated by Pearson Chi-square test.Data of serum AMH, Il-1A and Klotho levels, follicular count and mRNA levels was evaluated by independent, two-sample two-tailed t-test for unequal sample sizes and unequal variances with significance of BRCA carriers may be potentially predisposed to enhanced aging.


Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) . Although varying levels of tolerance have been achieved through conventional breeding, to date, effective resistance to CBSD within East African cassava germplasm has not been identified. RNAi technology was utilized to integrate CBSD resistance into the Ugandan farmer-preferred cassava cultivar TME 204. Transgenic plant lines were generated expressing an inverted repeat construct (p5001) derived from coat-protein (CP) sequences of CBSV and UCBSV fused in tandem. Northern blots using probes specific for each CP sequence were performed to characterize 169 independent transgenic lines for accumulation of CP-derived siRNAs. Transgenic plant lines accumulating low, medium and high levels of siRNAs were bud graft challenged with the virulent CBSV Naliendele isolate alone or in combination with UCBSV. Resistance to CBSD in the greenhouse directly correlated to levels of CP-derived siRNAs as determined by visual assessment of leaf and storage root symptoms, and RT-PCR diagnosis for presence of the pathogens. Low expressing lines were found to be susceptible to CBSV and UCBSV, while medium to high accumulating plant lines were resistant to both virus species. Absence of detectable virus in the best performing p5001 transgenic lines was further confirmed by back-inoculation via sap or graft challenge to CBSD susceptible Nicotiana benthamiana and cassava cultivar 60444, respectively. Data presented shows robust resistance of transgenic p5001 TME 204 lines to both CBSV and UCBSV under greenhouse conditions. Levels of resistance correlated directly with levels of transgene derived siRNA expression such that the latter can be used as predictor of resistance to CBSD.Cassava brown streak disease (CBSD) threatens food and economic security for smallholder farmers throughout East and Central Africa, and poses a threat to cassava production in West Africa. CBSD is caused by two whitefly-transmitted virus species:  Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV)  and cassava brown streak disease (CBSD). Distribution of CBSD is presently restricted mostly to eastern and central Africa. However, CBSD is spreading, and now presents a significant threat to cassava production in West Africa, including Nigeria, the world’s largest cassava producing country . CBSD is (UCBSV) , both ofa tabaci . Yield la tabaci , with rea tabaci . CBSD caa tabaci , most faa tabaci , but sucNicotiana benthamiana (Ugandan cassava brown streak virus (UCBSV-CP) and Cassava brown streak virus (CBSV-CP) fused in tandem  promoter  , regeneration of transgenic p5001 TME 204 lines and estimation of T-DNA copy number have been reported by The present study utilized an RNAi construct consisting of an inverted repeat of near full-length CP genes cloned from CBSV isolate Naliendele  (GenBank HG965221 position 7944-8839) and UCBSV isolate (UCBSV[UG:T04-42:04]) (GenBank HG965222 position 7892-8790). The CP sequences were fused in tandem and the constitutive promoter  cloned apromoter  is based, Japan) . The p50in vitro-grown plantlets using TRIzol Reagent . Thirty micrograms of total RNA was separated on 15% CriterionTM TBE-Urea Precast Gels  and transferred to HybondTM N+ membrane  using a semi-dry trans blotter . RNA probes for hybridization were prepared using a digoxigenin (DIG) RNA labeling kit with SP6/T7 polymerase according to manufacturer instructions . Two probes were generated, one each to detect siRNAs specific to CBSV and UCBSV. The partial CP gene sequences of CBSV and UCBSV  used to generate the inverted repeat sequence were cloned into the pGEM-T easy vector  and utilized as templates for in vitro transcription. The probes were hydrolyzed before use. Pre-hybridization of the membrane and hybridization of the probes with membrane bound RNA was performed at 40°C. Membrane washing, detection with CDP-star  and development of signal using HyperFilm ECL  were performed per manufacturer instructions.Transgenic TME 204 plants identified by Dot blot analysis to carry 1–2 copies of the T-DNA  were anaEach RNA sample was run and blotted twice, once for detection of siRNAs specific to the CBSV-CP and the other for UCBSV-CP using respective probes. Signal strength was determined by scanning the blots, followed by quantification using ImageJ software v. 1.50g (Rasband 1997-2015). Each Northern blot included control RNA extracted from plant line 718-01 of cultivar 60444 transgenic for construct p718 that consists of an inverted repeat construct from the CP sequence of UCBSV . TME 204In vitro micropropagated TME 204 plants transgenic for p5001 were transferred to soil  and 2 μg of the resulting total RNA reverse transcribed following the Invitrogen SuperScript® III First-Strand cDNA synthesis system per manufacturer instructions .Leaf samples were collected during the course of the experiment and storage root samples at the time of harvest, 12–22 weeks after graft inoculation. Storage root slices were frozen immediately in liquid nitrogen, freeze dried, and ground to a fine powder using a mortar and pestle. Samples of symptomatic and asymptomatic leaves were placed into 2 ml screw cap tubes and immediately frozen in liquid nitrogen. Ground storage root samples and leaf samples were homogenized in a FastPrep-24 machine  within 2 ml screw cap tubes, at settings of 4.0 M/S twice each for 30 s in presence of 700 μl of extraction buffer. For RNA extraction, 100–150 mg fresh weight leaf tissue and approximately 50 mg dry weight storage root tissue was used. RNA isolation was performed following the cetyltrimethylammonium bromide (CTAB) protocol . GenomicDetection of CBSV and UCBSV was carried out by RT-PCR as described previously by N. benthamiana. Procedures employed for sap inoculation of N. benthamiana followed those described previously  Naliendele isolate   and UCBSAgrobacterium-mediated transformation of cultivar TME 204 . Similarly, probes derived from CBSV-CP detected siRNA in transgenic plants transformed with p5001 and p5003 (plants expressing a CBSV-CP inverted repeat) .Approximately 450 independent transgenic cassava plants were recovered after  TME 204 . Of thes TME 204  and siRN repeat) , and froin vitro leaves of transgenic p5001 TME 204 plants was assayed by blotting once with the CBSV-CP specific probe, and once with the UCBSV-CP specific probe. Signal intensity was quantified by scanning Northern blots and analysis with ImageJ software. All blots included positive control RNA extracted from plant line 718-01 of cultivar 60444 transgenic for construct p718 ; low in 5001-199 (20% of the positive control); and medium to high in lines p5001-22 (48% of the positive control), 5001-173 (68% of the positive control), and 5001-207 (80% of the positive control).siRNA accumulation within uct p718 . This pluct p718 . In addiFigure 3). Quantification of signal intensities  indicated a strong positive correlation  between siRNA accumulation levels specific to the CBSV-CP and UCBSV-CP sequences within the same transgenic plant line . Therefore, for any given transgenic plant line, equivalent levels of siRNA accumulation were seen derived from the two CP sequence components present within the p5001 inverted repeat expression cassette.Analysis across all 169 p5001 lines revealed that 57 lines (34%) showed no detectable accumulation of siRNA, 40 lines (24%) showed relatively low expression level (1% -33% of the positive control) and 71 (42%) of the transgenic plant lines accumulated medium to high levels of siRNA (at ≥ 33.3% of the positive control) .Efficacy of p5001 CP-derived siRNAs for generating resistance to CBSD was assessed by bud graft inoculation of greenhouse-grown TME 204 plants. Wild-type plants of cultivar 60444, confirmed to be infected with CBSV or UCBSV, were used as the source of inoculum . ExperimWhen challenged with CBSV, CBSD leaf symptoms were first observed on non-transgenic TME 204 plants 7–15 days after graft inoculation. These were apparent as small chlorotic spots spread over the entire lamina of leaves about 10 nodes above the graft union , and subFigure 4) developed CBSD symptoms on leaves and/or stems by 9 weeks after challenge. Plant lines 5001-02 and 5001-03 were also classed as low expressers of transgene-derived siRNAs but accumulated more than that seen in 5001-04 . No CBSD symptoms were observed on stems or leaves across 9–10 clonal replicates of plant lines 5001-02 or 5001-03 by 9 weeks after bud graft inoculation. These plants were then cut back to 5 nodes above the initial graft and CBSD symptom development assessed on the newly formed leaves. Thirty percent of clonal replicates from line 5001-02 and 22% of 5001-03 developed leaf and/or stem CBSD symptoms over the subsequent 3 weeks. Likewise, the medium level siRNA accumulating line 5001-06 developed no CBSD symptoms on shoot tissues by 9 weeks after graft inoculation, with 27% developing leaf, but no stem, symptoms after cutback at 12 weeks after graft inoculation. All plants of the medium-high siRNA accumulating lines 5001-01, 5001-05, 5001-07, 5001-08, and 5001-09 , remained free of CBSD symptoms before and after cutback throughout the 12-week observation period.Cassava brown streak disease symptom development on stems and leaves was negatively correlated with levels of transgenic CP-derived siRNAs. Fifty percent of the clonal replicates of plant line p5001-04 that accumulated the lowest levels of siRNA . The frequency of storage roots within each line that displayed CBSD symptoms was also similar, ranging from 58–100% . Conversely, 8% of clonal replicate plants of the medium siRNA accumulating line 5001-06 possessed storage roots displaying CBSD symptoms, with only three out of 45 roots harvested from this RNAi plant line showing root necrosis. Average CBSD symptom severity (scores 1–5) across all symptomatic lines was comparable and ranged from 3.6–4.6 . As seen for shoot symptoms, all 146 storage roots harvested and examined from medium to high level siRNA expressing p5001 lines  were found to be asymptomatic for presence of CBSD . Therefore, occurrence of CBSD storage root symptoms was strongly and inversely correlated with the levels of transgene CP-derived siRNA accumulation as determined before virus challenge  . Transgenic plants were first inoculated with an axillary bud obtained from source plants infected with UCBSV, followed 14 days later by graft inoculation with a bud infected with CBSV , one medium (5001-06) and two high (5001-05 and 5001-07) siRNA accumulating events . All (100%) plants of the two low siRNA-expressing p5001 lines (5001-03 and 5001-04) showed CBSD storage root necrosis compared to 90% of non-transgenic TME 204 plants and 80% of 60444 line 718–801. In the medium siRNA-accumulating line 5001-06, 70% of the plants displayed CBSD storage root symptoms . The number of storage roots within each plant line showing CBSD necrosis was 95–100% for 5001-03 and 5001-04, 42% in 5001-06, 52% in 718-01 and 76% for wild-type TME 204, with average CBSD severity scores of symptomatic lines varying from 2.7 to 4.2 . In contrast, all plants of transgenic lines 5001-05 and 5001-07 were asymptomatic, with no disease observed across 20 (10 for each line) clonal replicates and a total of 60 harvested storage roots .At 21 weeks after completion of dual graft challenge, plants were harvested and storage roots evaluated visually for CBSD. Presence of CBSD symptoms in storage roots followed a similar pattern to that seen on plants challenged with CBSV alone . In the medium siRNA accumulating line 5001-06, only 25% of the clonal replicate plants tested positive for presence of CBSV, while all plants of the medium-high siRNA-accumulating lines  had no detectible CBSV in the sampled leaf tissues .The youngest fully expanded leaves were sampled from plants challenged with CBSV at 9 weeks after bud grafting and assayed by RT-PCR for presence of the virus. Presence of CBSV was confirmed in 80% of non-transgenic TME 204 plants and 67% of 60444 control line 718-01. Within the low siRNA-accumulating lines , CBSV was detected in 80, 54, and 83% of the plants tested, respectively . In the control line 718-01, 90% of the plants tested were positive for presence of CBSV, but all were found to be free of UCBSV. In the low siRNA accumulating line 5001-03, 60% of the clonal replicates were seen to be infected with both viruses, with the remaining 40% showing presence of CBSV only, while the medium level accumulating line 5001-06 had 60% of its plants positive for presence of CBSV, with the remaining plants free of detectable UCBSV or CBSV . All plants of the transgenic lines 5001-05 and 5001-07 showed no detectable levels of CBSV and/or UCBSV within their storage root tissue .Storage roots were analyzed by RT-PCR from plants co-inoculated with both CBSD causal pathogens using primers specific to the CP of each virus. All plants of wild-type non-transgenic TME 204 plants and the susceptible line 5001-04 were found to be positive for presence of both CBSV and UCBSV . In contrast, all 60444 plants graft inoculated with buds obtained from high siRNA-expressing lines 5001-05 and 5001-07 previously challenged with CBSV and UCBSV  remained free of CBSD symptom on leaves or stems . Similarly, N. benthamiana plants inoculated with sap extracted from leaf samples prepared from high siRNA-accumulating and CBSD-asymptomatic lines 5001-05 and 5001-07 failed to develop leaf symptoms. This contrasted with plants inoculated with sap extracted from CBSD-challenged wild-type TME 204 and susceptible lines 5001-04. In this case, all N. benthamiana plants developed typical CBSD symptoms characterized by stunting and curled leaves within 2–4 weeks of inoculation .Between 90 and 100% of 60444 plants inoculated from buds excised from infected donor plants developed CBSD symptoms on their leaves within 11–18 weeks . RT-PCR analysis of the storage roots from plants of 718-01 co-inoculated with UCBSV and CBSV confirmed the presence of CBSV, but not UCBSV , indicating that these plants remained resistant to the homologous virus species. Differences between field and greenhouse data demonstrated by line 718-01 could be explained by greater stringency of graft versus whitefly-mediated transmission of the ipomoviruses and/or the virulent nature of the CBSV Naliendele isolate . Figures 5 and 6). Plants from all five lines were also found to be free of detectable levels of CBSV and UCBSV based on RT-PCR diagnosis . Absence of CBSD pathogens within these TME 204 transgenic plant lines was further demonstrated by back-inoculation to CBSD susceptible N. benthamiana and cassava plants .In this study, plant line 718-01, which displayed field-level resistance to both virus species , succumb isolate  used in Figure 4). This confirms previous findings for RNAi-mediated resistance to CBSD in N. benthamiana and cassava cultivar 60444  in a manner similar to line 718-01. The enhanced protection conferred by the p5001 construct compared to p718 suggests that siRNAs derived from CP genes representing the two virus species are significantly superior compared to UCBSV alone for imparting durable protection against CBSD.Levels of resistance to CBSD were strongly and positively correlated with expression of CP-derived siRNA. The five transgenic lines that displayed resistance all accumulated medium-high levels of transgene-derived siRNA from both CP  would demonstrate effective resistance to CBSD. Absence of detectable siRNA expression in approximately one third of the lines (34%) and varying expression across the remaining transgenic p5001 TME 204 lines is most likely due to T-DNA integration position effects common for hylation . Based ohylation .We previously reported unexpected loss of inherent CMD2-mediated resistance to cassava mosaic disease in transgenic p5001 lines of TME 204 . The losNT, CF, TA, DM, and GB conceived the project. GB, NT, HW, and MI designed the project, carried out the experiments, generated and analyzed the data. RC and NT generated transgenic plants. GB and NT wrote the manuscript. All contributed and agreed to the final content.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.


Nivolumab, an immune checkpoint inhibitor, is recently clinically applied to non-small cell lung cancer (NSCLC) treatment, and this causes T cell activation and T cell infiltration to tumor tissue through the blockade of the interaction between programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1). 18F-fluorodeoxyglucose-positron emission tomography (FDG-PET) sometimes shows false positive because of the recruitment of neutrophils, lymphocytes, and macrophages. To date, there is only one report except our case, which described the correlation between FDG-PET and nivolumab.We report on a 75-year-old man on nivolumab treatment for metastatic non-small cell lung cancer. He had undergone right lower lobectomy for lung adenocarcinoma in the right S8 segment 10 months prior to recurrence. Pathological findings revealed invasive adenocarcinoma, pT1bN2M0 stage IIIA. Epidermal growth factor receptor (EGFR) mutation was positive for de novo T790M and anaplastic lymphoma kinase (ALK) rearrangement was negative. Immunohistochemistry was negative for PD-L1. He underwent chemotherapy with a combination of cisplatin and pemetrexed for four cycles but developed progressive disease involving the right hemithorax, multiple lymph nodes, and multiple osseous sites. Nivolumab was instituted as a second-line chemotherapy. After six courses of this immunotherapy, FDG-PET scan showed decreased FDG uptake in each recurrent lesion despite T lymphocyte activation by nivolumab. Serum carcinoembryonic antigen (CEA) level was also remarkably decreased.Nivolumab’s effect on recurrent NSCLC may be monitored by PET; larger studies are needed. Lung cancer is the leading cause of cancer-related death worldwide . Advance18F-fluorodeoxyglucose-positron emission tomography (FDG-PET) is an effective tool for assessing treatment response and surveillance of disease recurrence; however, it sometimes shows increased FDG uptake at sites of local inflammatory changes caused by the recruitment of neutrophils, lymphocytes, and macrophages . We presA 75-year-old man was admitted to our hospital because of metastatic NSCLC. He had undergone right lower lobectomy for lung adenocarcinoma originating in the right S8 segment as initial treatment. Postoperative findings revealed invasive adenocarcinoma with pT1bN2M0 stage IIIA. An EGFR mutation was positive for de novo T790M, and ALK rearrangement was negative. Immunohistochemistry was negative for programmed death ligand 1 (PD-L1)  after initial administration of nivolumab.In patients with recurrent or advanced NSCLC, who have relapsed after previous platinum-based chemotherapy or EGFR-TKI, docetaxel monotherapy is considered to be the current standard treatment regimen , 7. In aThe combination of FDG-PET and computed tomography (CT) is an effective tool for assessing treatment response and for surveillance for disease recurrence in cases treated with chemotherapy or radiation. Many investigators have reported the utility of FDG-PET or FDG-PET/CT in evaluating therapeutic response prospectively or retrospectively –20. CompThe optimal follow-up schedule of FDG-PET scan during immunotherapy is unclear. In fact, little is known about the role of FDG-PET and CT scan as appropriate radiological modalities. Some exploratory clinical studies (UMIN000020707 and UMIN000020814) have been conducted and ongoing in Japan to evaluate FDG-PET in nivolumab therapy. In our case, FDG-PET was considered to be a useful tool for the evaluation during immunotherapy. However, it is uncertain what the optimal timing of FDG-PET scanning will yield the highest specificity and sensitivity. More research in this area is needed.An urgent issue to be resolved is to explore more specific biomarker of this treatment because the correlation between PD-L1 expression by the tumor tissue and treatment effect with nivolumab has not been quantified. However, immunotherapy shows promise to improve the prognosis of patients with unresectable or recurrent NSCLC.We have found decreased FDG uptake by tumor after six courses of nivolumab treatment, suggesting the potential value of FDG-PET in monitoring the response to this immunotherapy. T cell activation and infiltration into tumor tissue did not affect the results of FDG-PET in this case. We await detailed results of ongoing clinical studies using FDG-PET for the evaluation of this immunotherapy.


Due to its high affinity for calcium phosphate, fetuin-A is highly abundant in mineralized bone matrix. Foreshortened femora in fetuin-A-deficient Ahsg-/- mice indicated a role for fetuin-A in bone formation. We studied early postnatal bone development in fetuin-A-deficient mice and discovered that femora from Ahsg-/- mice exhibited severely displaced distal epiphyses and deformed growth plates, similar to the human disease slipped capital femoral epiphysis (SCFE). The growth plate slippage occurred in 70% of Ahsg-/- mice of both sexes around three weeks postnatal. At this time point, mice weaned and rapidly gained weight and mobility. Epiphysis slippage never occurred in wildtype and heterozygous Ahsg+/- mice. Homozygous fetuin-A-deficient Ahsg-/- mice and, to a lesser degree, heterozygous Ahsg+/- mice showed lesions separating the proliferative zone from the hypertrophic zone of the growth plate. The hypertrophic growth plate cartilage in long bones from Ahsg-/- mice was significantly elongated and V-shaped until three weeks of age and thus prior to the slippage. Genome-wide transcriptome analysis of laser-dissected distal femoral growth plates from 13-day-old Ahsg-/- mice revealed a JAK-STAT-mediated inflammatory response including a 550-fold induction of the chemokine Cxcl9. At this stage, vascularization of the elongated growth plates was impaired, which was visualized by immunofluorescence staining. Thus, fetuin-A-deficient mice may serve as a rodent model of growth plate pathologies including SCFE and inflammatory cartilage degradation.Fetuin-A / α The Ahsg+/+, Ahsg+/- or Ahsg-/- mice. The fact that both immunostaining and Cibersort were negative for T cells suggests that the interferon type gene regulatory network is growth plate-intrinsic, and may be a salient part of growth plate physiology. This interferon type gene regulatory network was strongly induced in chondrocytes in fetuin-A-deficient mice.IFNɣ is mainly produced by natural killer (NK) cells and T cells . We probAhsg-/- growth plates. The regulation of STAT1 expression occurs through a positive feedback loop and its up-regulation strongly indicates the activation of the JAK-STAT signaling pathway [Ahsg+/+, Ahsg+/- and Ahsg-/- mice of different ages  in  pathway . Using aent ages . HypertrCXC-Ligand 9 (Cxcl9) was the most highly induced gene in the growth plates of Ahsg-/- mice, with an induction of 558-fold. CXCL9 was recently identified to counteract VEGF in bone vascularization, but has never been reported in growth plate cartilage [Cxcl9 in growth plates of 13-day-old Ahsg-/- mice, the elongated hypertrophic zones might be a consequence of deficient growth plate vascularization. Therefore we stained the vasculature in 13-day-old mice on thick histological sections. Ahsg-/- mice was indeed reduced compared to Ahsg+/+ mice, suggesting a role for CXCL9 in the initial stages of the growth plate dysplasia in Ahsg-/- mice.The chemokine artilage . As we fAhsg-/- mice have shortened femoral bones, indicating a crucial role for fetuin-A in endochondral ossification.Fetuin-A is a major non-collagen protein in bone, but its exact role in bone physiology is unclear. Fetuin-A-deficient Ahsg-/- mice. We noticed that the distal femoral epiphysis and the growth plate were both severely deformed, indicating a slippage of the growth plate during endochondral ossification, a phenotype similar to the human disease slipped capital femoral epiphysis (SCFE).Here, we studied the role of fetuin-A in endochondral ossification by investigating femur development and gene expression in growth plate chondrocytes. Computed tomography enabled a 3-dimensional and detailed visualization of the foreshortened femora from Ahsg-/- distal femoral growth plates, we found an unusually strong induction of inflammatory genes, especially genes regulated by IFNɣ. Cxcl9 was the most strongly induced (558-fold) gene in Ahsg-/- growth plates. The chemokine CXCL9 is expressed during inflammation and promotes the chemotaxis of pro-inflammatory cells, especially Th1 lymphocytes [Ahsg-/-, Ahsg+/- and Ahsg+/+ growth plates. Furthermore, we confirmed increased STAT1 expression and phosphorylation in Ahsg-/- and Ahsg+/- growth plate chondrocytes compared to wildtype. Taken together, our results suggest that JAK-STAT signaling is growth plate chondrocyte-intrinsic, especially during the hypertrophic state. In the absence of fetuin-A, this physiological JAK-STAT interferon type gene regulatory network is strongly induced resulting in e.g. the 558-fold induction of Cxcl9. We hypothesize that hypertrophic chondrocyte-derived CXCL9 may contribute to physiological cartilage remodeling by recruiting remodeling cells. Furthermore, diminished calcified matrix metabolism in the absence of fetuin-A may lead to an exaggerated Cxcl9 induction causing excessive remodeling cell activation and lesion formation in the growth plate instead of physiological remodeling.In phocytes ,34. TherAhsg-/- mice had elongated V-shaped hypertrophic zones. A recent publication demonstrated that the chemokine CXCL9 inhibits angiogenesis through direct antagonization of VEGF [Ahsg-/- mice was indeed diminished. Thus, the elongated hypertrophic zone in Ahsg-/- mice most likely resulted from a lack of vascular invasion caused by excessive Cxcl9 expression in the growth plate. Similarly, mouse models with a deficiency in either cartilage remodeling or vascular invasion  have likewise elongated hypertrophic zones, attesting to the close interplay of vascularization and tissue remodeling during endochondral ossification [Our results showed that until about three weeks postnatal, growth plates from  of VEGF . Staininfication –37.Ahsg-/- and Ahsg+/- mice showed lesion-associated cells in their growth plates, located between the proliferative and the hypertrophic zone at three weeks of age. Decreased safranin O staining indicated cartilage degradation around these cells. It is presently unclear, if these cells represent chondrocytic cells degrading matrix, or if they represent infiltrating bone marrow cells causing the lesion.Both, Ahsg expression in growth plates. These data suggest that, in addition to the fetuin-A protein released by the liver, the fetuin-A protein expressed locally by hypertrophic chondrocytes might also contribute to growth plate development. Therefore, deletion of Ahsg in hypertrophic chondrocytes might partially contribute to the growth plate defects observed in fetuin-A-deficient mice. Further studies involving liver- or growth plate-specific deletion of Ahsg may allow the dissection of the roles of fetuin-A derived from each source in the normal growth plate development.Immunofluorescent staining demonstrated that fetuin-A was predominantly localized in the cytoplasm of hypertrophic chondrocytes. Consistently, qRT-PCR showed a very low level of Ahsg-/- mice is comparable to the human disease slipped capital femoral epiphysis (SCFE) [The slippage of the distal femoral growth plate in s (SCFE) , which fAhsg-/- mice well before any lesions had formed, we suggest that inflammatory tissue remodeling may in fact trigger the mechanical weakening and slippage of the growth plate in SCFE patients.SCFE is the most common hip disorder in adolescents, with an incidence of 0.2 to 10 per 100,000 per year ,39. BiomAhsg+/- mice which produce about half the amount of fetuin-A compared to their Ahsg+/+ littermates also had lesions in their growth plates. Fetuin-A is a negative acute phase protein, thus its blood levels markedly decrease upon systemic inflammation [Fetuin-A itself may be involved in SCFE development. Heterozygous ammation . TherefoOur results suggest that the absence of fetuin-A elicits an inflammatory response in growth plate chondrocytes. The exact molecular events leading to the expression of IFNɣ-responsive genes in the growth plate and the subsequent changes in gene expression, growth plate slippage and dysplasia will be the subject of further studies.S1 FigAhsg+/+ and Ahsg-/- mice as shown in this figure.Angles were measured on contrast-inverted 2D sagittal cross-sections from μCT measurements of bones from eight-week-old (TIF)Click here for additional data file.S2 FigAhsg-/- and eight-week-old Ahsg+/- mice staining negative for cell surface markers CD45, F4/80 and CD3. (F-J) show bone marrow cells of the same bone sections staining positive for CD45, F4/80 and CD3, suggesting that the lesion-associated cells were neither hematopoietic cells, macrophages, or T cells. (K-O) Diff-Quick histology of lesion-associated cells in growth plate lesions from four-week-old and eight-week-old mice  were compared to blood smears from wildtype mice (M-O), suggesting that lesions at both ages had only mononuclear cells ruling out the presence of osteoclasts and granulocytes.Histological sections were prepared from paraffin-embedded decalcified bone samples. (A-E) illustrate lesion-associated cells in growth plates of four-week-old (TIF)Click here for additional data file.S3 FigSafranin O/fast green staining of decalcified paraffin sections from 13-day-old mice show elongated and V-shaped hypertrophic zones in the growth plates of long bones.(TIF)Click here for additional data file.S4 FigMice were injected with EdU, sacrificed after 2 h and EdU (seen as green nuclei) was visualized on decalcified histological sections.(TIF)Click here for additional data file.S5 FigAhsg+/+, Ahsg+/- and Ahsg-/- mice, were manually dissected (n = 4 mice per group). Additionally, Ahsg+/+ liver samples were taken (n = 4). RNA was isolated, reverse transcribed and Ahsg mRNA expression was analyzed using qRT-PCR. Fold-changes were determined using the ΔΔCt method, using Ahsg+/+ liver as control. Expression values were compared to liver expression values using One-Way ANOVA: ***p<0.001. N.D. = not detectable.Growth plates, adjacent bone tissue and the remaining epiphyses from distal femora and proximal tibiae from 13-day-old (TIF)Click here for additional data file.S1 TablePrimers were designed using NCBI primer-BLAST, or taken from Primer Bank. We ensured that all primers were spanning exon-exon junctions and primed the coding region of the gene.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table2 of the mean raw spot intensity after normalizing the microarray expression data. The raw spot intensities were averaged over biological replicates. Values for Ahsg+/+ and Ahsg-/- liver expression were taken from an independent microarray analysis using the same platform. Note that the spot intensities for growth plates of all genotypes and for Ahsg-/- liver were similarly low at around 25, while the wildtype liver spot intensity was 214.Gene expression is given as the log(DOCX)Click here for additional data file.S4 TableAhsg+/+, Ahsg+/- and Ahsg-/- mice. Pearson correlation coefficient, root mean square deviation (MSD) and p-value confirmed the validity of the analysis.Cibersort assumes that the gene expression data is derived from hematopoietic cells and estimates the fraction of each type of immune cell for each sample by comparing the gene expression data to established signatures of different immune cell subsets. The fractional value for 22 types of immune cells including seven T cell subtypes is listed in each row, thus for each the values add up to 1. Cibersort indicated that the fractions of T cell subtypes in the growth plates were uniformly low with no differences in growth plates derived from (DOCX)Click here for additional data file.


In contrast to other materials, UiO-66(Zr)-2COOH demonstrated a superior adsorption performance to verapamil due to their strong acid-base and/or hydrogen-bond interactions, and the adsorption process fitted well with the pseudo-second-order kinetic model. As verapamil-adsorbed materials were used for desorption experiments, ZrO2 demonstrated the most favorable desorption performance, whereas UiO-66(Zr)-2COOH yielded the poorest desorption capability. These Zr-based materials had also been coated at the surface with filter papers for the analysis of various drugs and proteins in the process of paper spray mass spectrometry. The results demonstrated that among the studied materials, ZrO2-coated paper gave the most favorable desorption performance as a pure drug solution, whereas the paper from UiO-66(Zr) demonstrated the optimal capability in the analyses of therapeutic drugs in a complex matrix  and a protein . The dynamic pore systems and high surface areas of flexible metal–organic framework materials make them excellent candidates to be used in different kinds of adsorption processes. However, the adsorption and desorption behaviors of therapeutic drugs on metal–organic frameworks in solution are not fully developed. Here, we systematically investigated the adsorption and desorption behaviors of a typical therapeutic drug, verapamil, over several Zr-based metal–organic frameworks [e.g., Zr-FUM, UiO-66(Zr), UiO-66(Zr)-NH Metal–organic frameworks (MOFs) ,2 have r2  as well as ZrO2 have been systematically investigated using paper spray mass spectrometry. The results demonstrated that in contrast to other materials, UiO-66(Zr)-2COOH presented a superior adsorption ability to the studied therapeutic drug verapamil due to their strong acid-base or hydrogen-bond interactions. As those materials were used for desorption experiments, ZrO2 showed the most favorable performance. In addition, we coated the investigated Zr-based materials onto the surface of filter paper and studied the elution or desorption behaviors of different drugs and proteins from the coated papers. ZrO2-coated paper was found to display the most favorable elution behaviors to studied drugs prepared in pure solvent, whereas UiO-66(Zr)-coated paper demonstrated special tolerance to a sample matrix, with the optimal performance to drugs in a complex matrix such as blood and protein analysis. The present study not only paves the way to explore the adsorption and desorption performances of various Zr-based materials with paper spray mass spectrometry, but also provides a deep insight into the application of metal–organic frameworks in analyses of different samples. In summary, the adsorption and desorption performances of various Zr-based materials [e.g., Zr-FUM, UiO-66(Zr), UiO-66(Zr)-NH


Oxidative stress is involved in pathophysiology and pathological conditions of numerous human diseases. Thus, understanding the mechanisms underlying the redox homeostasis in cells and organs is valuable for discovery of therapeutic drugs for oxidative stress-related diseases. Recently, by applying chemical biology approach with an ARE activator, BTZO-1, we found macrophage migration inhibitory factor (MIF) as a new regulator of antioxidant response element- (ARE-) mediated gene transcription. BTZO-1 and its active derivatives bound to MIF and protected cells and organs from oxidative insults via ARE activation in animal models with oxidative stress such as ischemia/reperfusion injury, inflammatory bowel diseases, and septic shock. In this review, we briefly highlight key findings in understanding the MIF-ARE system. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) play a dual role, beneficial and deleterious functions, in living systems , 2. ROS γ-glutamylcysteine synthetase (γ-GCS), and peroxiredoxin 1 . It. It32]. Clinical development of ARE activators as therapeutic drugs is an active area. For example, dimethyl fumarate (DMF) (Tecfidera™), the effects of which are believed, at least in part, to be mediated via Nrf2-ARE system, has been approved by the U.S. Food and Drug Administration as a therapy for multiple sclerosis . CDDO-MeHere, we propose that MIF could be a critical regulator of ARE-mediated gene expression. Quite interestingly, BTZO-1 activated the ARE-mediated gene expression under oxidative stress conditions without showing cytotoxicity. This unique profile of BTZO-1 may open the door for the discovery of new and safer ARE activators as therapeutic drugs for multiple disorders. Drug screening campaigns using radio-labeled BTZO-1 and MIF protein are worth trying to identify novel MIF binders with potential to activate ARE-mediated transcription. Further studies using BTZO-1 derivatives will be needed to narrow down the best indications for ARE activators using the MIF-ARE system.


This review article summarizes the recent progress in dental applications of natural products. Catechin gel showed selective antimicrobial activity, whereas the alkaline extract of various plant species rich in lignin carbohydrate complex (LCC) showed much higher antiviral activity than lower molecular weight polyphenols. Mouthwash with the alkaline extract of a plant classified as OTC effectively reduced halitosis. Unexpectedly, many polyphenolic compounds purified from the natural kingdom showed much lower tumor-specificity against human oral squamous cell lines as compared with antitumor agents, although they showed apoptosis-inducing activity. The alkaline extract of bamboo leaf, which exerted various common biological activities with LCC, showed osteogenic activity by stimulating differentiation toward osteoblasts while inhibiting differentiation toward osteoclasts. LCC enhanced the dectin-2 mRNA expression in macrophages, whereas glucan showed anti-osteoblastic action via dectin-1. These data suggest that natural products exert their biological activity by interacting with these molecules. It is generally accepted that many natural products effectively reduce oxidative stress and show chemoprevention activity in cell cultures and preclinical animal models ,2,3. HowPolyphenols present in the natural kingdom are roughly classified into the following three groups: tannins, flavonoids, and lignin-carbohydrate complexes (LCC) . TanninsFlavonoids are secondary metabolites synthesized from chalcones and categorized into flavonols, flavones, flavanones, isoflavones, pterocarpan, and coumestan. Resveratrol is classified as a stilbenoid.p-coumaryl, p-conifery, and sinapyl alchohols. Some polysaccharides in the cell walls of lignified plants are linked to lignin, and recovered as LCC after extraction with an alkaline solution.Lignins are formed through phenolic oxidative coupling processes. Lignin macromolecules are formed by the dehydrogenative polymerization of three monolignols: The oral cavity contains nearly half of the commensal bacterial population of the human body. An increase in the number of these microorganisms may produce systemic diseases such as infective endocarditis and aspiration pneumonia as well as oral infections. In order to suppress the onset of diseases, it is important to control the total numbers of these microorganisms. Green tea catechin showed a bactericidal effect against Gram-negative anaerobic rods, and the slow-release, local delivery of catechin combined with mechanical treatment improved periodontal status .Actinomyces, periodontopathic bacteria, and certain tested Candida strains, possibly due to the produced hydrogen peroxide, while it did not inhibit the growth of the oral streptococci that are important in the normal oral flora -6-methoxy-4H-1-benzopyran-4-one , (E)-3-[2-(4-chlorophenyl)ethenyl]-7-methoxy-2H-1-benzopyran  showed the highest tumor-specificity with the least keratinocyte toxicity   LCC isolated from pine cone extract appeared in the blood of mice after 3 h, and thereafter was excreted into the urine and feces . Since L


Arabidopsis thaliana, Brassica rapa, and certain crops.At high vegetation density, shade-intolerant plants sense a reduction in the red (660 nm) to far-red (730 nm) light ratio (R/FR) in addition to a general reduction in light intensity. These light signals trigger a spectrum of morphological changes manifested by growth of stem-like tissue  instead of harvestable organs —namely, shade avoidance syndrome (SAS). Common phenotypical changes related to SAS are changes in leaf hyponasty, an increase in hypocotyl and internode elongation and extended petioles. Prolonged shade exposure leads to early flowering, less branching, increased susceptibility to insect herbivory, and decreased seed yield. Thus, shade avoidance significantly impacts on agronomic traits. Many genetic and molecular studies have revealed that phytochromes, cryptochromes and UVR8 (UV-B photoreceptor protein) monitor the changes in light intensity under shade and regulate the stability or activity of phytochrome-interacting factors (PIFs). PIF-governed modulation of the expression of auxin biosynthesis, transporter and signaling genes is the major driver for shade-induced hypocotyl elongation. Besides auxin, gibberellins, brassinosteroids, and ethylene are also required for shade-induced hypocotyl or petiole elongation growth. In leaves, accumulated auxin stimulates cytokinin oxidase expression to break down cytokinins and inhibit leaf growth. In the young buds, shade light promotes the accumulation of abscisic acid to repress branching. Shade light also represses jasmonate- and salicylic acid-induced defense responses to balance resource allocation between growth and defense. Here we will summarize recent findings relating to such hormonal regulation in SAS in  Over the past few decades, a substantial body of studies has focused on understanding how plants sense the proximity of neighbors, how they respond at molecular levels, and how they adjust their morphological and physiological indexes. Many important light signaling components have been shown to regulate the shade avoidance responses—for example, PIFs (phytochrome interacting factors), HFR1 (long hypocotyl in far-red 1), PAR1/2 (phytochrome rapidly regulated 1/2) and COP1 (constitutive photomorphogenic 1). Meanwhile, various phytohormones are also involved and coordinated to shape shade-regulated plant architecture. Analyses of hormonal biosynthetic and signaling mutants, combined with studies of exogenous hormone applications, have implicated the roles of these phytohormones in multiple shade avoidance responses. In this review, we provide an overview of the current understanding of shade light and subsequent hormonal regulation.Light-quality signals are of paramount importance in detecting neighboring vegetation. Photosynthetic pigments in leaves absorb strongly in the range of photosynthetically active radiation (PAR) (400–700 nm) and UV radiation (280–400 nm), and reflect far-red wavelength (700–800 nm) . Thus, nArabidopsis phyB mutant plants indicates that phyB plays a dominant role in inhibiting SAS (Phytochromes exist in two photoconvertible forms: an inactive R-absorbing Pr form and an active FR-absorbing Pfr form. The steady-state ratio of Pr and Pfr forms depends on R/FR. The constitutive shade avoidance syndrome (SAS) phenotype of ting SAS . High R/ting SAS .To prevent exaggerated shade-avoidance responses, shade-induced HFR1 , PAR1/2 Cryptochromes (CRYs) are involved in repressing a low blue-mediated SAS by regulating PIF abundance and activity . PIF actUV-B-mediated inhibition of shade responses has been reported to occur through the degradation of PIF4/5 .In summary, downstream of photoreceptors, PIFs, as the key regulators, determine the massive transcriptional reprogramming upon perception of shade light, and also mediates the convergence between light and hormones.Arabidopsis identified TAA1, an enzyme catalyzing the first step of an auxin biosynthetic pathway (YUCCA (YUC) genes has been functionally positioned as the second and rate-limiting step of TAA1-dependent auxin biosynthesis . The transcriptional regulation of YUCCA genes by PIF7 has been found to link photoperception with auxin biosynthesis  exhibits an impaired shade-induced hypocotyl elongation  epicotyls  concentration in bean internode , cowpea picotyls , sunflowpicotyls  and Arabseedling . The shactive GA .gai gain-of-function mutant, which has a stable GAI (DELLA) protein, shows a reduced SAS  because the BR biosynthesis mutant dwarf1  and rot3f1  and BES1-INTERACTING MYC-LIKE (BIM) are positive regulators of SAS hypocotyl responses because ed shade . Remarkaed shade . The traed shade . Given ted shade . In conced shade . Althouged shade .In particular, the BR response appears to be required for the full expression of the SAS phenotypes under low blue light . The queThe role of cytokinins (CKs) in shade avoidance responses was discovered from the response of plants to vertical light intensity gradients in leaf canopies . In shadAtCKX6 (cytokinin oxidase) in the incipient vein cells of developing primordia  phyB inactivation led the plants to produce a blend of JA-induced monoterpenes that increased their attractiveness to the predatory mirid bug Macrolophus pygmaeus , a subunit of Mediator, is required for both JA-dependent defense gene expression and shade-induced early flowering . These fSalicylic acid (SA)-dependent disease resistance is also reduced under shade, which is considered as the early warning signal for plant competition . ReducedHelianthus annuus) (NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3) and NCED5, particularly in hypocotyls  are up-regulated by a neighbor signal (Abscisic acid (ABA) is commonly known as the “stress hormone” that responds to a variety of environmental stresses including both biotic and abiotic stress. Shade conditions increase ABA levels in sunflower ( annuus)  and toma annuus) . Shade ipocotyls . Severalr signal .nced3-2 and aba2-1) exhibited enhanced branching capacity under low R/FR.Shade light exerts a strong influence on branch development . One recHowever, ABA was not involved in shade-induced petiole elongation , suggestArabidopsis, BRC1 (BRANCHED1) is up-regulated in the axillary buds of plants grown at high density and is required for shade-mediated branch suppression  is one of the hormones that control lateral shoot growth. In pression . In sorg in buds . The invArabidopsis max2 mutants show longer hypocotyls under red, far-red and blue light than wild–type plants . The regulations of these phytohormones on the SAS described here might vary according to tissue type (This review focused on understanding the interaction between phytohormones and the SAS (sue type , stage osue type  and specsue type . In thisCY and LL designed and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


These double-bond-containing triblock copolymers are stable under UV irradiation and free radical circumstances.A series of ABA triblock copolymers, consisting of double-bond-containing poly (PPOA), poly(methyl methacrylate) (PMMA), or poly(butyl acrylate) (PBA) segments, were synthesized by sequential free radical polymerization and atom transfer radical polymerization (ATRP). A new bifunctional initiator bearing azo and halogen-containing ATRP initiating groups was first prepared followed by initiating conventional free radical homopolymerization of phenoxyallene with cumulated double bond to give a PPOA-based macroinitiator with ATRP initiating groups at both ends. Next, PMMA- The presence of any unique polymeric structure will definitely influence the properties of polymer since the nature of polymer rests with its structure. Polyallene, which can be obtained by the selective polymerization of either part of cumulated double bonds of allene derivatives, is expected to provide many idiographic properties because of its unique structure of possessing reactive exomethylene or substituted exomethylene directly linked to the backbone. This kind of reactive polymer has been intensively investigated because they can be utilized as attractive synthetic precursors for functional materials due to the versatility of addition reactions of double bondset al. developed Ni-catalyzed living coordination polymerization of allene derivatives to provide well-defined polymers with controlled molecular weights and narrow molecular weight distributions in high yields under mild conditions using [NiOCOCF3]2 as initiator4567871014Allene derivatives are often regarded as the isomers of propargyl derivatives since they possess cumulated double bonds and they can be polymerized by many different approaches such as free radical, cationic, coordination, and zwitterionic polymerizations using [η-allylNiOη3-ally)NiOCOCF3]2. Although random radical copolymerization of allene derivatives and common vinyl monomers could easily introduce the reactive double bonds into the conventional polymers, the molecular weights of the obtained copolymers are very difficult to be controlled and the molecular weight distributions are generally broadHowever, facile preparation of block copolymers from allene derivatives and common vinyl monomers including styrene and (meth)acrylate still remains a challenge because the polymerizations of usual vinyl monomers can not be initiated by ) vs. time, which exhibited the linear dependence of the conversion of BA on the extending of polymerization time, i.e. first order polymerization kinetics. This point distinctly demonstrated the typical character of ATRPThe conversions of BA monomer and the molecular weights of the resultants were determined by GC and GPC, respectively, so as to study the polymerization kinetics of bulk ATRP of BA. b-PPOA-b-PBA 4 triblock copolymer was examined by 1H NMR. 1H NMR spectrum of PBA-b-PPOA-b-PBA 4 triblock copolymer, which displayed all typical proton resonance signals of PPOA and PBA blocks. The peaks at 0.76, 0.93 (‘j’), 1.37 (‘i’), 1.59, 1.89 (‘h’), 2.27 (‘g’), and 4.05 (‘e’) were ascribed to all 12 protons of BA repeated unit, i.e. CH2CHCO2CH2CH2CH2CH3, respectively. Meanwhile, the double bonds in PPOA segment kept inert during ATRP of BA since their proton resonance signals around 5.10 ppm were still visible after the polymerization of BA monomer. Thus, we can conclude from the aforementioned evidences that double-bond- containing triblock copolymers comprising polyallene-based block and polyacrylate-based PBA segment, i.e. PBA-b-PPOA-b-PBA 4 triblock copolymers, were successfully constructed via Br-PPOA-Br 2 initiated ATRP of BA.The chemical structure of PBA-2 macroinitiator and PMMA-b-PPOA-b-PMMA 3d triblock copolymer under radical circumstance.Since most of crosslinking and degradation of common polymers under atmosphere follow the radical mechanism and the as-prepared polyallene-based triblock copolymers bear labile double bonds in PPOA block, we therefore examined the stability of Br-PPOA-Br 2 macroinitiator, it was exposed to UV irradiation plus benzophenone (photo-sensitizer) or AIBN  at ambient temperature  as listed in More importantly, PMMA-b-PPOA-b-PMMA and PBA-b-PPOA-b-PBA triblock copolymers by the combination of free radical polymerization, ATRP and the site transformation strategy, employing a bifunctional initiator possessing azo and Br-containing ATRP initiating groups as starting material. Polyallene-based macroinitiator was prepared by free radical polymerization of phenoxyallene initiated by the bifunctional initiator, which contains two ATRP initiating groups at both ends evidenced by elemental analysis and GPC/MALS. The target triblock copolymers were obtained by ATRP of MMA and BA initiated by the macroinitiator, respectively; and the polymerization process showed first order kinetics. This class of triblock copolymer is the first report of block copolymer via allene derivative and (meth)acrylate. The application of these double-bond-containing triblock copolymers is being explored in our group since they showed the exciting stability under radical circumstance, either with UV irradiation or with heat.We have displayed the detailed synthesis of PMMA-It is clear that the development of the as-prepared bifunctional initiator will make a significant contribution to the synthesis of polyallene-based block copolymers because PMMA and PBA segments in the current case can be easily extended to various hydrophilic and hydrophobic polymers for yielding diverse hydrophobic and amphiphilic block copolymers bearing double-bond-containing polyallene block.How to cite this article: Ding, A. et al. Double-bond-containing polyallene-based triblock copolymers via phenoxyallene and (meth)acrylate. Sci. Rep.7, 43706; doi: 10.1038/srep43706 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Airway epithelium forms a barrier to the outside world and has a crucial role in susceptibility to viral infections. Cyclic adenosine monophosphate (cAMP) is an important second messenger acting via two intracellular signaling molecules: protein kinase A (PKA) and the guanidine nucleotide exchange factor, Epac. We sought to investigate effects of increased cAMP level on the disruption of model airway epithelial barrier caused by RSV infection and the molecular mechanisms underlying cAMP actions. Human bronchial epithelial cells were infected with RSV-A2 and treated with either cAMP releasing agent, forskolin, or cAMP analogs. Structure and functions of the Apical Junctional Complex (AJC) were evaluated by measuring transepithelial electrical resistance and permeability to FITC-dextran, and determining localization of AJC proteins by confocal microscopy. Increased intracellular cAMP level significantly attenuated RSV-induced disassembly of AJC. These barrier-protective effects of cAMP were due to the activation of PKA signaling and did not involve Epac activity. Increased cAMP level reduced RSV-induced reorganization of the actin cytoskeleton, including apical accumulation of an essential actin-binding protein, cortactin, and inhibited expression of the RSV F protein. These barrier-protective and antiviral-function of cAMP signaling were evident even when cAMP level was increased after the onset of RSV infection. Taken together, our study demonstrates that cAMP/PKA signaling attenuated RSV-induced disruption of structure and functions of the model airway epithelial barrier by mechanisms involving the stabilization of epithelial junctions and inhibition of viral biogenesis. Improving our understanding of the mechanisms involved in RSV-induced epithelial dysfunction and viral pathogenesis will help to develop novel anti-viral therapeutic approaches. PCDH1 (Protocadherin 1 gene) and CDHR3 (encoding cadherin-related family member 3) with asthma [The airway epithelial barrier functions as the front line of host defense against airborne threats. The integrity of this barrier is essential for the regulation of innate immunity of the lungs, but such barrier integrity is known to be disrupted by a number of environmental stimuli. Barrier properties of the airway epithelium are primarily determined by the Apical Junctional Complex (AJC) composed of tight junctions (TJ) and adherens junctions (AJ) . It has h asthma , as wellh asthma . While vh asthma , mountinh asthma . These fRespiratory syncytial virus (RSV) is the most common respiratory virus that causes lower respiratory tract infection and inflammation. Historically considered a pediatric disease, RSV infection is now recognized as an important source of morbidity and mortality in elderly and high-risk adults . InvestiThe pathogenic mechanisms of RSV infection remain poorly understood. Specifically, little is known about the effects of RSV on the structure and functions of the airway epithelial barrier. Some previous studies , 20 demoColeus forskoliii plant and it is known to activate adenyl cyclase, increasing the level of intracellular cAMP. The efficacy of oral forskolin for asthma prophylaxis was trialed during two small, single-center studies, and found to reduce asthma attacks, which was thought to act through cAMP smooth muscle relaxation [Cyclic adenosine monophosphate (cAMP) is an important second messenger required for many critical homeostatic cellular functions . cAMP islaxation , 30. Howlaxation –33.There are very few studies on the effect of cAMP signaling in airway epithelial cells, and more importantly, the effect of cAMP signaling on RSV-infected epithelium has not been investigated. The goal of this study was to test the hypothesis that an elevated cAMP level could prevent AJC disruption and increased permeability caused by RSV infection in model airway epithelial cell monolayers. We report a barrier-protective role for cAMP in the infected epithelial cells that involves multiple mechanisms, such as stabilization of AJ and TJ structure, attenuation of RSV-induced rearrangement of the cortical cytoskeleton, and inhibition of viral propagation.The following primary monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) were used to detect junctional and signaling proteins by immunofluorescence labeling and immunoblotting: anti-occludin, anti–ZO-1, and anti–E-cadherin mAb ; anti–β-catenin mAb , Anti-claudin 1 and 4 pAbs , anti-cortactin mAb , anti-cortactin pAb (H222), anti-phospho-CREB  rabbit mAb, and anti-CREB (48H2) rabbit mAb , anti-GAPDH mAb . Fluorescently labeled phalloidin 488 (Thermo-Fisher Scientific) was used to visualize actin filaments. Anti-rabbit and anti-mouse secondary antibodies conjugated to Alexa-488 or Alexa-568 dyes were obtained from Thermo-Fisher Scientific. Mouse and rabbit secondary HRP-conjugated antibodies were purchased from GE Healthcare .Forskolin and H-89 were obtained from Sigma-Aldrich ; 8-Bromo-cAMP and 8CPT-2Me-cAMP were purchased from Tocris Bioscience . High-molecular-weight polyI:C . Fluorescein-conjugated 3-kDa dextran was obtained from Thermo-Fisher Scientific.2 insert and grown in defined media  with Ham’s F12) and differentiated at the air-liquid interface as evidence by completely dry apical surfaces, and by transepithelial electrical resistance greater than 1500 Ω/cm2 surface area, observed ciliary movement, and mucus production [16HBE14o- human bronchial epithelial cells  were cultured in collagen-coated transwells or 24-well plastic plates as previously described , 34. PriWild-type RSV strain A2 (RSV A2) stocks were grown as previously described . Polariz2 were used in these experiments. The presented data are calculated as percent changes compared to either vehicle-treated controls or time zero of viral infection.Epithelial permeability to small ions was evaluated by transepithelial electrical resistance (TEER) measurements of normal and RSV-infected cell monolayers using an EVOMX voltohmmeter . Only well-differentiated cell monolayers with TEER >500 Ω x cmEpithelial permeability for larger molecules was evaluated by measuring transmonolayer fluxes of fluorescein isothiocyanate (FITC)-conjugated dextran 3 kDa (Invitrogen), as previously described , 36, 37.Epithelial cell monolayers grown on transwell inserts were subjected to different fixation protocol. To label the AJC proteins and cortactin, cells were fixed with either 100% cold methanol or ethanol, respectively. To visualize actin filaments and CREB proteins, the cells were fixed in 4% neutral-buffered paraformaldehyde (PFA), with subsequent permeabilization with 0.05% Triton-X100. The fixed cells were incubated with specific primary antibodies, followed by incubation with Alexa Fluor-labeled secondary antibodies. Nuclei were stained with DAPI (Sigma-Aldrich).Immunofluorescently-labeled cell monolayers were examined using an Olympus FluoView 1000 confocal microscope  with a 100X U Plan S Apo 1.4 NA oil objective. The Alexa Fluor 488 and 568 signals were imaged sequentially in frame-interlace mode to eliminate cross talk between channels. The images were processed using Olympus FV10-ASW 2.0 Viewer software and Adobe Photoshop. Images shown are representative of at least 3 independent experiments, with multiple images taken per slide.After indicated treatments of cell monolayers in cell culture plates, cells were washed with cold PBS and lysed in RIPA buffer . Protein concentration was quantified with a bicinchoninic acid (BCA) protein assay . Equal concentrations of protein from each sample were resolved on a SDS-PAGE and transferred to a PVDF membrane . Membranes were blocked with non-fat dry milk, or in cases of phosphor antibodies with bovine serum albumin (BSA), and probed with primary antibodies overnight followed by secondary antibodies. Membranes were exposed to ECL  and protein bands were detected using X-ray film. GAPDH was used as a lane protein loading control.5’ CACCCTGTTGGAAAC 3’ and 5’ CTCTGTCAGTTCTTG 3’-from Sigma-Aldrich). Transcript expression was normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the housekeeping gene. The relative change in gene expression was calculated using the formula: % change = 2^-(ΔΔCt) = 2-ΔCt(treated samples)- ΔCt(control samples) where ΔCt = -Ct (detected gene)-Ct(GAPDH) and Ct is the threshold number.Total RNA was extracted from pretreated monolayers in cell-culture plates using E.Z.N.A total RNA kit  with additional DNase digestion (Omega). cDNA was synthesized using qScript cDNA Synthesis Kit  and was amplified by real-time PCR with an iQ5 multicolor Real-Time PCR Detection System (Bio-Rad) using SYBR Green fastmix (Quanta Bioscience) and primers targeting the 87 bp sequence of the RSV strain A2 genome, which encodes for the viral fusion (F) protein  as per manufacturer’s instructions.P value of less than 0.05.Data were analyzed using Prism software  and Microsoft Excel. Data are representative of three or more experiments and are presented as means ± SEMs. Data were evaluated statistically with ANOVA or the Student’s t-test, with Bonferroni correction for multiple comparisons. Significance was considered at a To test the hypothesis that increased intracellular cAMP level could protect the epithelial barrier from disruption caused by RSV infection, polarized airway epithelial cells were infected with RSV strain A2 at a 0.5 MOI in the presence or absence of forskolin, a known activator of adenylyl cyclase, which is responsible for cAMP production. In agreement with our previously published data , RSV cauin vitro [16HBE14o- cells have been shown to exhibit well-defined AJCs and appear morphologically similar to airway epithelial cells in vitro , 38, 39.Given the fact that forskolin is a potent inductor of cAMP production, we next examined whether the protective effects of forskolin on airway epithelium is associated with an increase in intracellular cAMP. Polarized bronchial epithelial cells were infected with RSV  for varying lengths of time (1–24 h), followed by stimulation with forskolin (20 μM) for 15 minutes. At all tested times, intracellular cAMP was increased by 30–40 fold from baseline after 15 minutes added forskolin to the RSV-infected cell cultures . Note thSince the observed barrier-stabilizing effects of forskolin correlated with the increased production of cAMP, we sought to determine if cAMP analogs could also play protective roles in RSV-infected cell monolayers. Furthermore, we asked if forskolin and cAMP-dependent stabilization of the airways epithelial barrier involve activation of its major effector molecule, Epac. In order to answer these questions, we used 8-Bromo-cAMP  and 8CPT-2Me-cAMP, which is a cell-permeable selective activator of Epac . A dose-Collectively, these results suggest that forskolin-induced elevation of intracellular cAMP attenuates RSV-induced disruption of the model airway epithelial barrier in an Epac-independent fashion.The lack of barrier-protective effect of the pharmacologic Epac activator suggests that forskolin and cAMP attenuate the effects of RSV on epithelial junctions by activating PKA. To test this suggestion, we measured the effects of forskolin on phosphorylation and activation (nuclear translocation) of CREB (cAMP response element-binding) protein, which is a known direct substrate of PKA . PolarizThe infectious cycle of RSV consists of attachment and entry into the host cell, transcription, replication, assembly, and release of new viral particles. During viral entry into epithelial cells, RSV F protein mediates fusion of the virus to the cell membrane, and subsequent cell-to-cell spread by fusion of neighboring cell membranes resulting in the formation of syncytia. Since both forskolin and 8-Bromo-cAMP decreased RSV-induced syncytia formation, we hypothesized that elevated intracellular cAMP, and its downstream signaling effectors, might decrease RSV F mRNA titer. To test this possibility, bronchial epithelial monolayers were infected with RSV  in the presence or absence of forskolin or cAMP analogs, and expression of a viral 87 bp sequence encoding the F protein was quantified by real-time PCR. We found that expression of RSV A2 F mRNA significantly decreased in epithelial cells pretreated with either forskolin or 8-Bromo-cAMP, while 8CPT-2Me-cAMP did not affect RSV A2 F expression . In a paTo gain additional insights into the effects of elevated cAMP on the viral infection, we infected cells with RSV , with subsequent addition of forskolin at different times following viral inoculation (0–24 h). Immunofluorescence labeling and confocal microscopy indicate that forskolin inhibited RSV-induced disruption of the AJC structure when it was added after RSV inoculation . InteresThe results obtained in this study emphasize two different effects of increased cAMP level in RSV-infected airway epithelial cells. One effect is the attenuation of virus-induced barrier breakdown and AJC disassembly, and the other effect is the inhibition of RSV F mRNA expression and viral propagation within the epithelial monolayers. This creates an important question whether the described barrier-protecting effects of cAMP represent an independent mechanism or is strictly a secondary effect of the inhibited viral propagation. To answer this question, we utilized a synthetic, non-replicating, double-stranded RNA viral mimic (dsRNA Polyinosinic:polycytidylic acid or polyI:C) –47. In aThe integrity and permeability of different epithelial barriers are regulated by the perijunctional actin cytoskeleton . Our preEmerging evidence indicates a critical role for the airway epithelial barrier in regulating responses to environmental stimuli such as allergens and viral infections , 50. Whiin vitro. This approach involves increasing intracellular cAMP level by either applying a pharmacological adenylyl cyclase activator, forskolin, or by using a stable cell-permeable analog of cAMP. The increased cAMP attenuated all detrimental effects of RSV infection on the epithelial barrier, which include increased permeability to ions and large non-charged tracers, as well as TJ and AJ disassembly  of actin filaments, since RSV infection also altered localization , and phoThe exact mechanisms underlying the PKA-dependent regulation of the actin cytoskeleton remain poorly understood, yet several scenarios can be envisioned. One scenario involves direct PKA-dependent phosphorylation of different actin-binding proteins. Examples of such PKA targets are vasodilator-stimulated phosphoprotein that elongates actin filaments  and alphThe pathophysiologic implications of RSV-induced airway epithelial barrier dysfunction are not well understood. Epidemiological studies have repeatedly shown an association between RSV infection, subsequent recurrent wheeze, and chronic airway inflammation. Studies have indicated that E-cadherin levels in asthmatic patients’ sputum correlate with asthma severity . Loss ofAnother important finding of this study is that an increase in cAMP level inhibits expression of RSV F protein and viral propagation in the airway epithelial cell monolayers Figs  & 5. RSVin vivo models of RSV infection will be essential to investigate the roles and mechanisms of viral-induced disruption of the pulmonary barrier in the complex physiology of the lungs.In summary, this study provides compelling evidence of cAMP/PKA-mediated protection against RSV-induced disruption of the model airway epithelial barrier. Our most striking findings are the multiple targets of cAMP actions in the infected epithelium that include the stabilization of the AJC and the inhibition of RSV propagation. Furthermore, these antiviral and barrier-protective functions of cAMP can be efficiently executed at different stages of the RSV infection, including late stages of RSV propagation within the epithelium. Scientific insights gained from these data will likely guide the design of future therapeutic approaches to treat acute and chronic sequelae of RSV infection. In addition, future studies are planned to further dissect the long-term effects of RSV-induced AJC disruption, persistent lung inflammation, and the role of cAMP signaling pathways. Ultimately, with regards to cAMP/PKA signaling effects, RSV is a major cause of lower respiratory tract infection and one of the primary reasons for hospitalization worldwide. There is no efficient antiviral therapy or vaccine to manage the disease. The cAMP signaling pathway is involved in the regulation of many essential cellular processes in differentiated epithelial layers. Here we sought to identify the effects of RSV on the integrity of the airway epithelial barrier and the modulation of these effects by cAMP signaling. We found that activation of cAMP prevents RSV-induced airway epithelial barrier dysfunction. Furthermore, cAMP activation decreased RSV viral titer even when treatment was delivered after RSV infection. These findings provide novel insights into understanding epithelial cell responses to a clinically significant virus with poorly defined pathogenesis and limited treatment options.


Pre-challenge NAb titers feasibly provide sterile protection from SIV/SHIV infection by purging the earliest onset of viral replication and likely modulate innate immune cell responses. Sufficient sub-sterile NAb titers after established infection also confer dose-dependent reduction of viremia, and in certain earlier time frames augment adaptive immune cell responses and even provide rebound-free viral control. Here, we provide an overview of the obtained patterns of SIV/SHIV protection and viral control by various types of NAb passive immunizations and discuss how these notions may be extrapolated to NAb-based clinical control of HIV infection.Neutralizing antibody (NAb) responses are promising immune effectors for control of human immunodeficiency virus (HIV) infection. Protective activity and mechanisms of immunodeficiency virus-specific NAbs have been increasingly scrutinized in animals infected with simian immunodeficiency virus (SIV), chimeric simian/human immunodeficiency virus (SHIV) and related viruses. Studies on such models have unraveled a previously underscored protective potential against  Normally, a combinational response of these two effectors, initiated and assisted by professional antigen-presenting cells (APCs) such as dendritic cells (DCs) and CD4+ helper T lymphocytes (Th), effectively kill and neutralize infected cells and cell-free virus, respectively, resulting in elimination of virus from the infected host.Viral infections overcoming the host sentinel hurdles of cell-intrinsic and innate immune responses are met with well-concerted adaptive immune responses. Adaptive immune responses comprise cellular and humoral effectors, and the central players for each are CD8+ (R5) memory CD4+ T cell-tropic (R5-tropic) human immunodeficiency virus (HIV) and pathogenic simian immunodeficiency virus (SIV) infections are met with inefficient adaptive immune responses, resulting in persistent viral replication  immunodeficiency viruses which propagate more dominantly Alternatively, it was also found that polyclonal neutralizing IgG existing at viral challenge can confer slower disease onset as well as enhanced survival in orally R5-SHIV-infected newborn macaques, suggestive of a lasting protective effect by preexisting NAb titers , potent anti-HIV bNAbs at high titers can also provide viremia suppression  cell-dependent mechanisms were also explanatory of the above non-sterile protective effects. Another line of work showed engineered Fc receptor-binding properties of bNAb VRC01 being related with extended bioavailability, altered localization (antibody transcytosis) and improved protection against SHIV challenge  and showed stimuli-specific metabolic quiescence, as defined by enrichment of a phosphorylated AMP kinase-low CTL subpopulation which is indicative of exhaustion-free T-cell qualitative preservation  passive polyclonal NAb infusion in SIV+/CD8+ T-cell priming. Alternatively, in another two-dose (days 1 plus 14) polyclonal NAb infusion model, cases of sterile protection and set-point viremia reduction were obtained in SIVsmE660-challenged macaques, and this associated with enhanced endogenous de novo NAb responses . Importantly, this pattern of synergism between endogenous adaptive immune cells and early short-term NAb administration has also become well-conceptualized in murine retrovirus-infected mice  antiretroviral therapy [(c)ART] is another important point. One work on the effect of acute-phase ART on SIV-infected rhesus macaques  administration of bNAbs , also resulted in significant 0.8–2.5 log10 reduction in viremia -specific bNAb KD-247 resulted in decreased viremia in HIV-infected patients, as well as an obtained case of ongoing viremia control after NAb decline  NAbs to be infused. For prophylactic vaccine induction of HIV-specific NAbs, in addition to the rational design of vaccine Env antigen, designing attainment of synergism with concomitantly induced T-cell responses may delineate protective responses much more potent than what is currently expected.NAbs, when present by passive immunization, are being recognized as capable of playing a central role in sterile protection against and post-infection control of SIV and SHIV infection. Future studies aiming for NAb immunotherapy-based HIV clinical control shall proceed with a pharmacological perspective on the HY conceived and drafted manuscript; TM co-drafted manuscript.This work was supported by Japan Agency for Medical Research and Development, the Ministry of Health, Labor, and Welfare and the Ministry of Education, Culture, Sports, Science, and Technology in Japan ([JSPS] KAKENHI).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.


Myeloid-derived suppressor cells (MDSCs), a heterogeneous population of cells composed of progenitors and precursors to myeloid cells, are deemed to participate in the development of tumor-favoring immunosuppressive microenvironment. Thus, the regulatory strategies targeting MDSCs' expansion, differentiation, accumulation and function could possibly be effective “weapons” in anti-tumor immunotherapies. Epigenetic mechanisms, which involve DNA modification, covalent histone modification and RNA interference, result in the heritable down-regulation or silencing of gene expression without a change in DNA sequences. Epigenetic modification of MDSC's functional plasticity leads to the remodeling of its characteristics, therefore reframing the microenvironment towards countering tumor growth and metastasis. This review summarized the pertinent findings on the DNA methylation, covalent histone modification, microRNAs and small interfering RNAs targeting MDSC in cancer genesis, progression and metastasis. The potentials as well as possible obstacles in translating into anti-cancer therapeutics were also discussed. Howevin vivo . One expin vivo , 6.+Ly6G+Ly6Clow cells, called G-MDSCs, are demonstrated to have a granulocytic phenotype and express high levels of reactive oxygen species (ROS) but only nominal amounts of nitric oxide (NO). G-MDSCs exert immunosuppressive function via ROS-mediated mechanisms in a cell contact dependent manner [+Ly6G-Ly6Chigh cells, called M-MDSCs, present a monocytic-like morphology and exert immunosuppressive function via high expression of inducible nitric oxide synthase (iNOS) and arginase-1 following the activation of STAT3 signaling in a cell contact independent manner [+CD33+HLA-DRlow/- cells [+CD14-CD11b+CD33+HLA-DRlow/- G-MDSCs and CD15-CD14+CD11b+CD33+HLA-DRlow/- M-MDSCs, but with no agreement to date [MDSCs are a heterogeneous population of cells generally composed of progenitors and precursors to dendritic cells, macrophages and granulocytes at various stages of differentiation , 8. In pt manner . To be st manner . In contt manner . The inct manner  and prevt manner . NO is at manner  or to int manner . Both oft manner -18. Unli/- cells . Some in to date .MDSCs are reported to involve in a large variety of disorders such as infectious diseases , inflammde novo and inherited DNA methylation is dependent on DNA methyltransferases (DNMTs) [de novo methylation[DNA methylation, one of the most important forms of epigenetic modification, inhibits gene expression with transcription machinery: Once DNA is methylated, transcriptional factors are blocked from gaining access to the gene, and thus expression is effectively silenced. Both  (DNMTs) , which t (DNMTs)  . To be thylation.Cannabis sativa plant, is validated to be potent inducers of MDSCs [Δ9-Tetrahydrocannabinol (THC), an exogenous cannabinoid derived from the of MDSCs . In factof MDSCs .Thereforof MDSCs . In addiof MDSCs .Covalent histone modification, another form of epigenetic regulation, refers to the process in which covalent bonds alter the core structure of histones and influence the binding of ‘effector’ molecules to DNA sequences, therefore affecting patterns of gene expression, 46. The+Ly6C+F4/80intCD115+) in vivo and in vitro, which suppressed allogeneic T cell proliferation in a NOS- and heme oxygenase (HO)-1-dependent manner [Histone acetylation and deacetylation modulate gene expression in a mutually antistatic way. In the process of histone deacetylation, histone deacetylases (HDACs) are recruited to the gene promoters by transcriptional factors or co-repressors, where they block gene accessibility and transcription. HDAC inhibition enables to increase the extent of histone acetylation, resulting in tighter DNA binding and reduction in gene expression . TSA is t manner .versus host disease (GVHD) [in vivo [HDAC11 is the newest member of the histone deacetylase family and has been reported to involve in hematopoietic lineage differentiation, as well as graft e (GVHD) . In the [in vivo . The tra[in vivo . Tumor-b[in vivo . ConsideIn cancer, G-MDSC is the dominant subpopulation of MDSC that accumulated in tumor microenvironment. It is iPost-transcriptional regulation is a highly conserved biological phenomenon, in which microRNAs (miRNAs) specifically recognize and degrade a homologous host mRNA, leading to the targeted gene being silenced. This prin vivo [Noman et al. demonstrated hypoxia-inducible factor-1a (HIF1α)-induced over expression of miR-210 potentiated MDSC's tumor-promoting function by increasing arginase activity and NO production . In tumoin vivo . These rin vitro study. MiR-9 performed on MDSCs differentiation by targeting runt-related transcription factor 1 (Runx1), an essential transcription factor in regulating MDSC differentiation and function [Tian et al. reported in their study that miR-9 could inhibit the differentiation and promote immunosuppressive function of MDSCs . They fofunction . In Lewifunction . This st+Gr-1+ MDSCs induced by THC in vivo and identified differentially expressed miRNA unique to functional MDSCs including up-regulation of miR-690, miR-22, miR-15b and miR-27a and down-regulation of miR-324-5p, miR-335-5p. Altered miRNA expression regulated the target transcription factors and genes involved in MDSC activation, expansion and myeloid maturation [Hegde and his colleagues observed THC led to robust induction of functional MDSCs in mice . They thturation . Especiaturation . MiR-690in vivo [Liu et al. identified that miR-494, whose expression was dramatically induced by TGF-β1, as an essential player in increasing the accumulation and activity of MDSCs by targeting of phosphatase and tensin homolog (PTEN) and activation of the Akt pathway . Expressin vivo . Suppresin vivo. Another study focused on the role of miR-155 in tumor promotion. It was illustrated that miR-155 accelerated the accumulation of functional MDSCs in the tumor microenvironment by suppressor of cytokine signaling (SOCS) 1 repression and reduced ability to license the generation of CD4+Foxp3+ regulatory T cells (Tregs), thereby facilitating tumor growth [Li et al. identified miR-155 and miR-21 as the two most upregulated miRNAs during the induction of MDSC from the bone marrow cells by GM-CSF and IL-6 . Overexpr growth . Host mi+ and CD8+ T cells both in vitro and in vivo [+CD11b+ cells isolated from the spleens of disease-free mice, indicating that tumor-associated factor downregulated the expression of these two microRNAs, which contribute to immune tolerant microenvironment in tumor sites [MDSCs transfected with miR-17-5p or miR-20a are less able to suppress Ag-specific CD4 in vivo . In tumoor sites .+Gr1+MDSCs in the presence of tumor-associated factors by targeting myocyte enhancer factor 2C (MEF2C) in vitro [+ Gr1+ MDSCs may exert an important role in controlling the increased accumulation of MDSCs in patients with tumor.Liu et al. demonstrated that miR-223 could remarkably inhibit differentiation of bone marrow cells (BMCs) into CD11bin vitro . In recoin vitro . Besidesin vitro . These sAlthough there were no evidences that miR-146a had direct influence on MDSCs' development, Boldin et al. reported that miR-146a, whose expression was up-regulated after immune cell maturation and/or activation, could inhibit the proliferation of multiple myeloid lineages, including CD11b+Gr-1+ population. In factIn humans, miR-424 up-regulation was associated with human monocyte/macrophage differentiation from CD34+ hematopoietic progenitors. The master transcription factorPU.1 activated the transcription of miR-424 and suppressed NFI-A, an inhibitor of monocyte differentiation, thereby enhancing M-CSFr expression and monocytic differentiation . Thus, uGarzon et al. studied the role of miRNAs in granulopoiesis in acute promyelocytic leukemia (APL) patients during all-trans-retinoic acid (ATRA) treatment . They foMiR-34a was able to induce MDSC expansion both in chimera and transgenic mice. Detailed study found that overexpression of miR-34a could inhibit MDSC apoptosis by suppressing the expression of N-myc but without affecting MDSC proliferation . This stSmall interfering RNAs (SiRNAs) are artificial double-strand RNAs (dsRNAs) of 21-25 nucleotides in length that generate during PTGS and RNAi . These sA20  is originally identified as a primary TNF-α responsive gene in human umbilical vein endothelial cells (HUVEC) . A20 gen+ T cells and these effects depended on reduced expression and enzymatic activity of arginase-1, a downstream STAT3 target gene and a potent T-cell inhibitor [In prostate cancer, the tumor-associated MDSCs potently inhibit autologous CD8+T cells proliferation and production of IFN-γ and granzyme-B, thereby impairing anti-tumor immunity. Hossain et al. previously generated an original strategy to silence genes specifically in toll-like receptor (TLR) 9 positive myeloid cells using CpG-siRNA conjugates and verified that human granulocytic MDSCs expressed TLR9 and rapidly internalized naked CpG-STAT3 siRNA, thereby silencing STAT3 expression , 80. Thenhibitor . Disruptnhibitor . Consistnhibitor . They fonhibitor . Thus, S+ Tregs, possibly suppressed Th2 responses and enhanced Th1 responses. Thus, SCF siRNA could improve immune therapy for the treatment of advanced tumors [It is acknowledged that tumor-derived factors are involved in the accumulation of MDSCs and blockade of tumor factors can prevent T-cell anergy and Treg development -85. Stemd tumors .in vitro and in vivo without displaying signs of toxicity [Caseine kinase 2 (CK2) is one member of serine/threonine kinase family. Despite of its constitutive activation and ubiquitous expression in a variety of cell types and tissues, its overexpression was documented in the number of solid and hematologic malignancies , 91. Chetoxicity . From thThe highlighted researches rendered supports to the epigenetic modulation of MDSCs by DNA and histone modification, microRNA and siRNA serving as effective immunotherapeutic strategies for fighting against cancer Table -2. TheseFirstly, these studies are mainly implemented in murine models of diseases since murine MDSCs have been explicitly identified while human MDSCs have not. Whether these epigenetic modulatory approaches could actually work on patients requires further researches on accurate identification of human MDSCs and pre-clinical studies. Secondly, recent discussions on immunoregulatory mechanisms have focused on whether MSDC-mediated T cell suppression is antigen-specific or nonspecific. We favor that MDSC's suppressive effect on T-cell responses is in an antigen-specific manner because of the finding that T cells in the peripheral lymphoid organs of tumor-bearing mice as well as in the peripheral blood of cancer patients can still respond to stimuli other than tumor associated antigens -95. Consin vivo, as discussed above, could probably be modulated by more than one miRNA. Do these miRNAs action in the sole or overlapping signaling pathways? Is it possible that a panel of miRNAs modulate different checkpoints of certain cell process in a synergistic way? These questions remain to be answered for that two or more genes can be knocked down or silenced simultaneously by using a miRNA cocktail regimen. For another, the assessment on pharmacodynamics and pharmacokinetics is indispensable to determine whether or not these reagents could be used as drugs. The safety property of these reagents should also be taken into consideration for severe adverse effects are not permitted regardless of the therapeutic effect. With breakthroughs regarding human MDSCs, we believe the epigenetic modification on MDSCs could benefit specific and effective treatment for cancers in time.For one thing, MDSC migration and function


Previous work has illustrated that blood flow patterns following aortic valve and/or aortic root replacement (AVR/ARR) are dependent on the valve and procedure type. Compared to healthy volunteers, ARR with bioprosthetic and mechanical valves demonstrated increased blood velocities and helicity/vorticity in the ascending aorta, with none restoring physiologic hemodynamics. On-X mechanical valves have unique design features, such as flared inlets and larger length-to-diameter ratios more analogous to the natural left ventricular outflow tract. Thus, we sought to examine the impact of this alternative design on ascending aortic hemodynamics.2), and aortic hemodynamics were visualized with streamlines (Ensight) and graded separately for helicity by two blinded reviewers . Patients' post-operative pressure gradients and helicity grades were then compared to pre-operative and control values via one-tailed t-tests and Wilcoxon signed ranks tests, respectively.11 patients  with aortic valve disease underwent contrast-enhanced 4D flow MR at 1.5T before and after AVR ± ARR with an On-X mechanical valve prosthesis. 11 age/gender-matched controls  were also analyzed to characterize baseline, physiologic flow patterns. 4D flow MR data were corrected for eddy currents, Maxwell terms, and velocity aliasing in Matlab. Peak transvalvular pressure gradients were computed from peak velocity, using the Simplified Bernoulli equation . Eccentric, helical flow was identified in all patients pre-operatively, which was significantly reduced following AVR ± ARR , restoring similar flow patterns to controls . Although not statistically significant, post-operative flow appeared supra-physiologic with absence of commonly-observed, right-handed, helical flow during systole in some patients (Figure Preliminary evidence suggests the On-X mechanical aortic valve design may restore physiologic hemodynamics in the ascending aorta in contrast to flow-patterns described in previous reports for other commercially available valve designs. The clinical implications of restoring near physiologic flow patterns after aortic valve surgery are unknown; however, less asymmetric distribution of wall shear stress may slow endothelial cell dysfunction and wall degeneration in the native aorta, reducing risk of future atherosclerotic change and aneurysm formation.