Myelophthisis due to melanoma is a rare phenomenon. Treatment strategies for patients with this serious complication of malignancy have not been well documented, and none have previously reported efficacy of immune checkpoint inhibition. Since bone metastases are not measurable lesions per standard response criteria, the efficacy of immune checkpoint inhibition in the bones is also not well described.We describe a patient with widespread melanoma metastases involving the bone marrow causing myelophthisis and pancytopenia who responded to immune checkpoint inhibition with the anti-programmed cell death-1 (PD-1) inhibitor pembrolizumab.This is the first report to our knowledge of disease response to immune checkpoint inhibition in a patient with myelophthisis. Clinical trials have recently emerged describing the efficacy of PD-1 inhibition for disorders regularly involving the bone marrow, such as hematologic malignancies, suggesting the importance of better understanding the bone marrow as an immunologically active compartment. Clinicians should be aware that immune checkpoint inhibition alone may be effective in treating malignancy involving the bone marrow, even in cases of extensive involvement resulting in pancytopenia due to myelophthisis from a solid tumor as our case suggests. Myelophthisis due to malignancy is a rare, but often significant, complication of cancer that occurs when metastases infiltrate bone marrow and impair normal hematopoiesis . AlthougWe describe the first case to our knowledge of a patient with widespread melanoma metastases involving the bone marrow causing myelophthisis who responded to immune checkpoint inhibition with the anti-programmed cell death-1 (PD-1) inhibitor pembrolizumab.An otherwise healthy 64 year old male presented in 2/2015 with a suspicious lesion on his back. Biopsy and subsequent wide local excision revealed a 1.2 mm deep, non-ulcerated cutaneous melanoma. A sentinel lymph node biopsy involving his right groin in 4/2015 revealed one lymph node involved with micrometastatic (<1 mm) melanoma. According to the 7th edition of the American Joint Committee on Cancer (AJCC) staging system, he had stage IIIA melanoma (pT2AN1A). A computerized tomography (CT) scan in 5/2015 was unremarkable for metastatic disease, and he elected to not proceed with a completion lymph node dissection. The patient considered adjuvant systemic treatment options, and he chose to proceed with radiographic surveillance alone. Basic laboratory testing including a complete blood count  was normal.Three months later, in 8/2015, the patient developed flu-like symptoms and fatigue with an accompanying low grade fever. He presented to the emergency room where blood work revealed new pancytopenia with a low white blood cell count of 3.1 (K/mcL) with a normal differential  and no immature cells. Hemoglobin was slightly low at 12.3 (g/dL), and platelets were low at 67 (K/mcL). He had no evidence of abnormal bleeding or bruising. A CT scan on 8/19/15 showed multiple lesions in liver, spleen, and some small pulmonary nodules consistent with metastatic disease. A liver biopsy confirmed metastatic melanoma, BRAF wildtype. Due to his pancytopenia, a bone marrow biopsy was additionally performed. Hematoxylin and eosin staining revealed extensive metastatic melanoma with decreased number of myeloid, erythroid and megakaryocyte lineages along with prominent melanophages Fig. . The melHe started systemic therapy with pembrolizumab on 9/10/15. Within 3 weeks of starting pembrolizumab, he began to feel better with improvements in fatigue and functional status. Laboratory results from 12/10/15 showed improvements in his pancytopenia  response criteria for assessment of therapy effectiveness . Since lThe emergence of immune checkpoint blockade against hematologic malignancies which often involve the bone marrow, such as Hodgkin lymphoma, has provided a glimpse into the potential benefits of these therapies beyond their initially described uses for solid tumors. Recent results from the KEYNOTE-013 study reporting the use of pembrolizumab for patients with classical Hodgkin lymphoma showed an overall response rate of 65% with the majority of those responses (70%) lasting longer than 24 weeks, all in a cohort of patients who were heavily pretreated and refractory to multiple lines of therapy . An addiOur report of a profound melanoma response within the bone marrow to pembrolizumab suggests the bone marrow is an immunologically active compartment. Yet, whether the mechanisms involved in antitumor immune responses to checkpoint inhibition within the bone marrow are similar to those at other sites of disease remains unknown. Some literature suggests receptor activator of nuclear factor-Kappa-B ligand (RANKL), which can be produced by osteoblasts, may be involved in influencing dendritic cells to enhance T regulatory cell immune attenuation . RANKL h


Due to the heterogeneity of cancer, identifying differentially methylated (DM) CpG sites between a set of cancer samples and a set of normal samples cannot tell us which patients have methylation aberrations in a particular DM CpG site.We firstly showed that the relative methylation-level orderings (RMOs) of CpG sites within individual normal lung tissues are highly stable but widely disrupted in lung adenocarcinoma tissues. This finding provides the basis of using the RankComp algorithm, previously developed for differential gene expression analysis at the individual level, to identify DM CpG sites in each cancer tissue compared with its own normal state. Briefly, through comparing with the highly stable normal RMOs predetermined in a large collection of samples for normal lung tissues, the algorithm finds those CpG sites whose hyper- or hypo-methylations may lead to the disrupted RMOs of CpG site pairs within a disease sample based on Fisher’s exact test.Evaluated in 59 lung adenocarcinoma tissues with paired adjacent normal tissues, RankComp reached an average precision of 94.26% for individual-level DM CpG sites. Then, after identifying DM CpG sites in each of the 539 lung adenocarcinoma samples from TCGA, we found five and 44 CpG sites hypermethylated and hypomethylated in above 90% of the disease samples, respectively. These findings were validated in 140 publicly available and eight additionally measured paired cancer-normal samples. Gene expression analysis revealed that four of the five genes, HOXA9, TAL1, ATP8A2, ENG and SPARCL1, each harboring one of the five frequently hypermethylated CpG sites within its promoters, were also frequently down-regulated in lung adenocarcinoma.The common DNA methylation aberrations in lung adenocarcinoma tissues may be important for lung adenocarcinoma diagnosis and therapy.The online version of this article (doi:10.1186/s12967-017-1122-y) contains supplementary material, which is available to authorized users. The incidence of lung adenocarcinoma is increasing worldwide. It is widely recognized that lung adenocarcinoma, like other cancers, has different molecular subtypes with different prognoses , 2. In cT test [Currently, genome-wide DNA methylation profiles are widely applied to identify population-level differentially methylated (DM) CpG sites in cancer tissues compared with normal controls using various statistical methods such as the Shannon entropy based method QDMR , empiricT test . HoweverT test , 13. HowRecently, we have reported an interesting biological phenomenon that the within-sample relative expression orderings of genes within a particular type of normal tissues are highly stable but widely disrupted in the corresponding cancer tissues . Based oIn this study, through the analysis of multiple methylation datasets for normal lung tissues, we firstly revealed an interesting biological phenomenon that the RMOs of CpG sites within different samples of normal lung tissues are highly stable but widely reversed in the cancer tissues. Based on this finding, we showed that the RankComp algorithm can accurately detect DM CpG sites in individual cancer samples from DNA methylation data for cancer samples alone. Then, RankComp was applied to identify DM CpG sites for each of the 539 lung adenocarcinoma samples from The Cancer Genome Atlas (TCGA). Many CpG sites with methylation aberrations in above 90% of lung adenocarcinoma tissues were found and validated in 140 publicly available and eight additionally measured paired cancer-normal samples. Gene expression analysis revealed that four of the five genes, HOXA9, TAL1, ATP8A2, ENG and SPARCL1, each harboring one of the five frequently hypermethylated CpG sites within its promoters, were also frequently down-regulated in lung adenocarcinoma.http://tcga-data.nci.nih.gov/tcga/). We used a dataset (GSE32861) of DNA methylation profiles for paired cancer and adjacent normal samples to evaluate the performance of RankComp (Table DNA methylation profiles for lung tissues were collected from the Gene Expression Omnibus (GEO)  databaset GSE3286 of DNA mt GSE3286 of DNA mThe DNA methylation data was measured with Illumina Human Methylation 27 Beadchip (27K array) and Illumina Human Methylation 450 Beadchip (450K array). We focused on analyzing the 25,978 CpG sites measured by both 27 and 450K arrays. Using methylated signal intensity (M) and unmethylated signal intensity (U), the DNA methylation level of each probe was calculated by M/(U + M + 100) . The proData of 234 pathways covering 5981 unique genes was downloaded from the Kyoto Encyclopedia of Genes and Genomes (KEGG) (Release 58.0)  for pathThe RMO of two CpG sites, A and B, was denoted as A > B or A < B if the site A had a higher or lower methylation level than the site B. The RMO of two CpG sites was defined as stable in normal tissues if the same RMO existed in at least 99% of the samples, allowing 1% detection error rate.s/k. k was the number of the stable CpG site pairs shared by the two lists, among which s pairs had the same RMO patterns (A > B or A < B) in the two lists. The probability of a concordance score s/k observed by chance was calculated according to the cumulative binomial distribution model [Pe (Pe = 0.5) is the probability of a pair of CpG sites having the same RMO in two lists by chance.For a particular type of normal tissues, we respectively detected two lists of stable CpG site pairs in two independent datasets to evaluate the reproducibility of the stable CpG site pairs. The concordance score of the two lists of stable CpG site pairs was calculated as on model .1\documeFor stable RMOs in normal samples, we applied Fisher’s exact test to detect gene pairs with significantly higher frequencies of reversal RMOs in cancer samples than what expected by random chance using two independent datasets, respectively, defined as reversal CpG site pairs. The p-values were adjusted using the Benjamini–Hochberg procedure . The conCi was differentially methylated in a given cancer sample by testing the null hypothesis that the proportion of reversal CpG site pairs supporting the hypermethylation of Ci was equal to the proportion of reversal CpG site pairs supporting the hypomethylation of Ci. For a given CpG site Ci, if its ordering was stably lower (or higher) than that of CpG site Cj in normal samples but this ordering was reversed in a cancer sample, then this reversal CpG site pair could support hypermethylation (or hypomethylation) of Ci in this sample. If Ci itself is not changed in methylation level, the effect of the methylation changes of other CpG sites on the upward or downward shift in the rank of Ci is assumed to be a random event.Then, we used RankComp to identify differentially methylated CpG sites in a given cancer sample in comparison with its previously normal state, based on the highly stable CpG site pairs with consistent RMOs in at least 99% of previously accumulated normal samples measured by different laboratories. The detail of the RankComp algorithm was described in Wang et al. . BrieflyMethylation profiles for paired cancer and adjacent normal tissues were downloaded from TCGA and GEO to evaluate the performance of RankComp. We used RankComp to detect DM CpG sites in individual cancer samples using DNA methylation data on cancer samples alone. After identifying DM CpG sites for each cancer sample, we evaluated the precision of DM CpG sites identified for this cancer sample using the observed methylation level differences (hyper- or hypomethylation) between the cancer sample and its paired adjacent normal sample as the golden standard. The underlying assumption of this evaluation is that the previously normal state of a cancer tissue could be approximately represented by the adjacent normal tissue of the cancer tissue. For a cancer sample, if the aberration states of DM CpG sites identified by RankComp are consistent with the golden standard, then they are defined as true positives (TP); otherwise, false positives (FP). The precision is calculated as positive predictive value: TP/(TP + FP).To ensure the association between the individualized CpG sites and cancer, the precision analysis for each cancer sample was restricted to the CpG sites that were found to be differentially methylated at the population-level. Thus, we applied T-test to detect population-level DM CpG sites between cancer samples and normal samples using two independent datasets for each cancer, respectively. The Benjamini–Hochberg procedure was used to control the false discovery rate (FDR). The concordance score between the two lists of DM CpG sites and its statistical significance were calculated by the same method as described above. We defined the DM CpG sites that were consistently detected from the two independent datasets as the population-level DM CpG sites for each cancer.Eight paired samples of lung adenocarcinoma tissues and adjacent normal tissues were used for DNA methylation and gene expression detection. Fresh frozen cancer tissue samples were obtained from surgically removed lung specimens. Tumor samples were macro dissected to ensure the purity of the tumor. From the same patient, adjacent normal tissue samples were collected from resected, located approximately 3.5–5 cm away from the tumor site. All samples were collected from the operating room immediately after surgical resection. Samples were fresh frozen and were shipped on ice for subsequent DNA extraction and RNA extraction. This study was approved by the institutional review boards of all participating institutions, and written consent forms were obtained from all participants.® Genomic DNA Mini Kit, as described by the manufacturer. DNA quantification was performed using a NanoDrop 2000 UV–Vis Spectrophotometer (Thermo). The bisulfite conversion of DNA was conducted using the Zymo bisulfite gold kit. The Infinium Methylation 450K assay was performed according to Illumina’s standard protocol. Processed methylation chips were scanned using an iScan reader (Illumina). All samples were processed at the same time to avoid chip-to-chip variation. Infinium Methylation data were processed using the Methylation Module of the GenomeStudio software package (v. 2011.1). For quality control, methylation measures with a detection p-value >0.05 were removed. The data was initially normalized using internal controls in the GenomeStudio software and has been submitted to GEO (GSE85845).DNA was extracted from frozen tissue samples using the QiagenTotal RNA was extracted using Trizol reagent (Invitrogen) according to the manufacture’s protocol. The purity and concentration of RNA was determined by Nano Drop ND-1000 spectrophotometer according to OD260/280 reading. Each time point has three replicates. Total RNAs were hybridized using mRNA + lncRNA Human Gene Expression Microarray V4.0 , which contains probes interrogating about 34,235 human mRNAs assembled from databases such as UCSC , RefSeq We collected a total of 355 samples for normal lung tissues, including 79 samples derived from three datasets assayed by the 27K array and 276 samples derived from two datasets assayed by the 450K array Table . Here, wp < 2.2 × 10–16). The stable CpG site pairs involved all the measured CpG sites. These results suggest that the highly stable within-sample RMOs of CpG sites in normal lung tissues can be reproducibly detected across samples measured with different platforms. As exemplified in Fig. We defined two CpG sites as a stable CpG site pair if the two CpG sites had identical RMO in at least 99% of the normal lung tissue samples. Accordingly, a list of 229,037,151 stable CpG site pairs was identified in the 79 normal lung tissue samples assayed by the 27K array, and a shorter list of 173,949,484 stable CpG site pairs was identified in the 276 normal lung tissue samples assayed by the 450K array  . These 4159 reproducible DM CpG sites were defined as the population-level DM CpG sites for lung cancer.Here, we firstly determined DM CpG sites associated with lung cancer at the population-level. From two independent datasets, GSE62948 and GSE32866, 6,515 and 5,451 DM CpG sites in lung cancer were detected  harboring frequently hypermethylated CpG sites within their promoters were also frequently down-regulated in lung adenocarcinoma. The results suggested that these genes might function as tumor suppressors and thus might be drug targets or diagnostic biomarkers of lung adenocarcinoma.In addition, after identifying DM genes for a disease sample, we could detect pathways significantly enriched with hypermethylated and hypomethylated genes respectively for this disease sample. The application of the individual-level pathway analysis to 539 samples of lung adenocarcinoma revealed that the neuroactive ligand-receptor interaction pathway, known to be involved in cancer development , 31, wasp < 0.0014, univariate Cox model) than the other patients without APC hypermethylation. This result supported a previous report that hypermethylation of APC could be associated with poor prognosis of NSCLC including lung adenocarcinoma [Notably, the majority of the DNA methylation aberrations are not universally appearing in patients of lung adenocarcinoma. For example, 83.64% of the CpG sites with DNA methylation aberrations appeared in less than 80% of all the 539 lung adenocarcinoma tissues from TCGA, reflecting the heterogeneity nature of lung adenocarcinoma. Some of these methylation aberrations might be cancer subtype specific and associated with distinct clinical outcomes. As a case study, we identified potential methylation aberration signatures correlated with the prognosis of stage I lung adenocarcinoma patients after surgical resection using all the 120 TCGA samples of stage I lung adenocarcinoma patients with surgical resection. For each CpG site, we classified the patients into two groups according to whether they had or had not the methylation aberration of this CpG site and then compared their relapse-free survival times after surgical resection . Finally, we found seven genes  that were significantly associated with the prognosis of patients. For instance, we found that APC was hypermethylated in 50% of the 120 patients and these patients had significantly shorter relapse-free survival time (arcinoma . Moreovearcinoma , PDLIM1 arcinoma , CAP2 [3arcinoma  and EDILIn this paper, we only analyzed the CpG sites within gene promoter regions, assayed by both the 27K and 450K arrays, but were unable to analyze the huge number of CpG sites located within gene bodies or intergenic regions due to the extensive computational burden. Thus, an optimized algorithm needs to be developed so that the genome-wide CpG sites assayed by 450K or whole-genome bisulfite sequencing could be taken into account. In addition, in order to extend the application scope of the RankComp algorithm, it is necessary to further evaluate the cross-platform properties of RMOs in samples measured by other platforms such as the whole-genome bisulfite sequencing platform.In summary, the individual-level analysis of DM CpG sites can help us identifying universal or subtype-specific DNA methylation biomarker for cancer prevention, treatment and diagnosis , 41.Using multiple DNA methylation datasets for normal lung tissues, we firstly revealed that the RMOs of CpG sites within different samples of normal lung tissues are highly stable but widely reversed in the corresponding cancer tissues. This biological phenomenon allows us to exploit the within-sample RMOs of CpG sites to accurately detect individual-level DM CpG sites using RankComp. Additionally, we used RankComp to identify DM CpG sites for each of the 539 lung adenocarcinoma samples from TCGA and identified many common DNA methylation aberrations in lung adenocarcinoma tissues, which were validated by paired cancer-normal samples. Using gene expression analysis, we further identified abnormal expression genes among genes with common DNA methylation aberrations. These common genes might be used as candidate biomarkers for lung adenocarcinoma diagnosis and therapy.


Low-dose aspirin is used worldwide for preventing thromboembolic disorders. Its use, however, is often associated with gastrointestinal bleeding, mostly due to direct irritation of the gastric mucosa. Here we provide evidence for a novel sublingual formulation of aspirin micronized and co-grinded with collagen proven to be as effective as oral standard formulation in inhibiting platelet aggregation but with attenuated gastric irritation. This represents a new option for better aspirin treatment in the prevention of myocardial infarction and stroke.,,,,Orally given low-dose aspirin has been used for decades due to its anti-inflammatory and antithrombotic properties.Recently, we developed and patented (N. 102015000079955) a new formulation of aspirin which leads to faster absorption and activity but devoid of direct gastrointestinal lesioning effect. In addition, this formulation allows sublingual administration of the drug which, by passing the liver metabolism, leads to faster serum peak concentration with more prominent and rapid inhibition of cycloxygenase (COX), the major target of antiplatelet and antinflammatory action of aspirin.Figure 1).Cristalline aspirin was amorphized via micronization and co-grinding with collagen (see 2 (TXB2), and its urinary metabolite 11-dehydro-TXB2 after both oral and sublingual standard as well as micronized aspirin were carried out to assess the inhibitory activity at the COX level.Micronized collagen co-grinded aspirin formulations were developed for both oral and sublingual administration  for 7 consecutive days produced a rapid rise of acetylsalicylic acid serum concentration which peaked at 2–4 h after the administration and declined 8–12 h later . This effect was accompanied by a decrease of TXB2 serum concentration at 6 h which declined 16–20 h after the administration. . A similar response was seen in healthy volunteers (n = 10) receiving 50 mg of standard aspirin orally, with lower effect compared to 100 mg, . No significant differences were seen when 50 and 100 mg of micronized and collagen co-grinded aspirin were given orally to healthy volunteers Oral administration of 100 mg of standard crystalline aspirin (n = 10 for each dose) of aspirin micronized and co-grinded with collagen, produced a dose-related peak of serum concentration of acetylsalicylic acid which occurred earlier compared to both sublingual and oral administration of aspirin (1 h) with a decline of serum concentration occurring 4–5 h after the administration . This effect was accompanied by early inhibition of TXB2 levels which was observed after 2 h and lasted 10 h after aspirin administration. Furthermore, the effect of aspirin formulation in TXB2 was confirmed by detection of its urinary metabolite11-dehydro-TXB2 after Day 7 of the study , thus suggesting that sublingual administration of micronized co-grinded aspirin displays a non-inferiority response on COX enzyme compared to crystalline standard formulation. Determination of serum TXB2 serum levels and of urinary 11-dehydro-TXB2 showed no changes before and after treatment in healthy volunteers receiving placebo (not shown).In contrast, sublingual administration of 50 and 100 mg (Neither changes on routine blood analytical biomarkers nor side effects or adverse drug reactions were noted in any of the groups after administration of oral or sublingual aspirin. Pill count adherence was 100% and no enrolled subject was excluded from the study see .Finally, to verify the attenuated impact on gastric mucosa of micronized collagen co-grinded aspirin compared to standard oral formulation, experiments have been carried out in rats receiving doses of aspirin proven to produce gastric lesions (see Our data show that sublingual formulation of aspirin micronized and co-grinded with collagen displays a better pharmakinetic profile compared with standard crystalline aspirin. This effect was accompanied by attenuated direct gastric ulcerogenic effect of the new formulation and by non-inferiority profile on TXB2 serum and urinary levels after a 7-day treatment compared to the standard formulation of aspirin. The potential remaining warning on gastrointestinal bleeding due to inhibition on prostaglandin-related production of protective gastric mucus of the new formulation is to be better clarified. This represents a new option for better aspirin treatment in the prevention of thromboembolic disorders.European Heart Journal—Cardiovascular Pharmacotherapy online.Conflict of interest: none declared.Supplementary MaterialClick here for additional data file.


To evaluate the aggressiveness of peripheral zone prostate cancer by correlating the Gleason score (GS) with the ratio of the diffusion tensor imaging (DTI) measures. Forty-two peripheral zone prostate tumors were imaged using DTI. Regions of interest focusing on the center of tumor foci and noncancerous tissue were used to extract statistical measures of mean diffusivity (MD) and fractional anisotroy (FA). Measure ratio was calculated by dividing tumor measure by noncancerous tissue measure.min) and the ratio of minimum MD (rMDmin) show the same highest correlation with GS (both ρ = −0.73). Between GS ≤ 7 (3 + 4) and GS ≥ 7 (4 + 3), differences are significant for all MD measures but for some FA measures. MD measures perform better than FA measures in discriminating GS ≥ 7 (4 + 3). Strong correlations are observable between GS and MD measures while weak correlations are present between GS and FA measures. Minimum tumor MD (MD Ratios of MD measures can be used in evaluation of peripheral zone prostate cancer aggressiveness; however tumor MD measures alone perform similarly. The clinical behavior of the prostate cancer is strongly correlated with Gleason score (GS) as high values of GS indicate more aggressive tumors and an increased risk of local and distant tumor spread . The preDWI employs three orthogonal gradient directions to capture diffusion signal quantified by an apparent diffusion coefficient (ADC) in the detection of cancerous prostate tumor foci and in the assessment of tumor aggressiveness. Recent studies report that the value of ADC is lower in the prostate tumor foci than the healthy prostate tissue and the tumor ADC is correlated with the tumor GS , 3. BeneCurrent clinical applications of DTI for prostate cancer are focused predominantly on differentiation of tumor from normal prostate gland –13 and sConsecutive patients with biopsy-proven prostate cancer who underwent MR imaging prior to the diagnosis of prostate cancer between December 2012 and December 2015 at our institute were considered for this retrospective study. Patients with benign findings or with transition zone prostate cancer or with peripheral zone prostate cancer but only having TRUS guided biopsy without complementary radical prostatectomy surgery were excluded. Patients who had peripheral zone prostate cancer proved with histopathology  and no contraindications to MRI were included. The institutional and research committee waived informed consent and approved this retrospective study.A total of thirty-eight patients aged 40–77 years  with forty-two identified prostate cancer tumors were taken into analysis, retrospectively . The tumor diameter was ranging from 5 to 55 mm . Twenty-four of the tumors had radical prostatectomy while the resting tumors had in-bore prostate biopsy. A detailed demographic data is presented in At our institute, prostate MR imaging has been conducted by a 3 T MR scanner  using a sixteen-channel phased array surface coil while patients were positioned still in a supine position. During imaging, to reduce motion artifacts due to bowel peristalsis, 20 mg of butylscopolamine  is administrated to the patient if tolerated. The imaging protocol includes T2-weighted imaging, dynamic contrast-enhanced imaging (DCE-MRI), and diffusion weighted imaging to validate mp-MRI. However, for the current study, the protocol is extended to incorporate diffusion tensor imaging (DTI). Average acquisition time for mp-MRI is 30 minutes and additional time for DTI is around 3 minutes.b-values (b = 0 and 800 s/mm2) with the following parameters: 77–81.3 ms TE, 3200 ms TR, 128 × 106 matrix size, 260 × 215 mm2 FOV, 3.6 mm slice thickness, and 1 × 1 × 3.6 mm3 interpolated voxel size.Triplanar T2-weighted images are acquired using a turbo spin-echo imaging sequence with 100 ms echo time (TE), 3566–3631 ms repetition time (TR), 512 × 352 matrix size, 200 mm field of view (FOV), and 3 mm slice thickness. DTI is performed in the axial plane using a 2D single shot echo-planar imaging sequence (EPI) in 12 gradient directions at two different Acquired images of mp-MRI are evaluated and each identified lesion is scored according to the PIRADS v2 classification system from ESUR. For a lesion having a PIRADS score ≥ 4, either in-bore prostate biopsy or TRUS guided prostate biopsy followed by radical prostatectomy is scheduled. The mean time interval between imaging and each procedure is 73 days and 34 days, respectively. In the case of radical prostatectomy, the specimens are fixed in 10% buffered neutral formalin and the surgical margins are painted with ink. Prostate is serially cut into 3-4 mm sections in a plane perpendicular to the long axis of the prostate (from apex to base). Each slice is sequentially submitted in total for routine tissue processing and as whole mount sectioning. Routine sections are stained with hematoxylin and eosin.All tumor foci are marked on 16-sector divided standardized prostate diagram by a pathologist. The pathologist is blind to any MRI interpretations and evaluates all tumor foci according to its Gleason score. In the case of multiple tumor foci separated by noncancerous tissue, each focus is considered separately. The GS of every foci is categorized into four risk groups: low (GS = 6), intermediate-low (GS = 7 (3 + 4)), intermediate-high (GS = 7 (4 + 3)), and high (GS = 8–10) risk cancers. Tumor sizes, PSA levels, and final tumor stages for those GS based groups are as reported in T2-weighted images and diffusion tensor images were transferred to a workstation for subsequent analysis. The images were evaluated by two radiologists  and a pathologist  using Syngo MR D13C software  installed on the workstation. T2-weighted images providing better anatomical orientation were used to guide the radiologists while localizing the tumors. When localized, a tumor was evaluated on the relevant MD map of DTI further to match the tumor focus considering the standardized histopathologic diagrams as reference while taking alterations in the prostate shape and size caused by preservation of the specimen into account. A tumor focus was assumed to be matched when it was in the same region on both histopathology and MRI. After matching, a region of interest (ROI) was manually placed on the relevant MD map with care to include only the center of tumor foci. An additional “reference” ROI with similar size as the tumor ROI was placed for the noncancerous tissue in the same prostatic region. All ROIs were circular with 3 mm diameter and placed by the two radiologists by mutual agreement. Both radiologists and the pathologist were blinded to Gleason score during image evaluation and ROI placement. Using the same software, the ROI pairs placed on the MD maps were automatically reproduced on the FA maps of DTI see .μ) and the standard deviation (σ) measures were used to assess contrast-to-noise ratio (CNR) for both MD and FA maps. The CNR was calculated by DTI measures determined by the software for each ROI were recorded. These measures included the minimum, the maximum, the mean, and the standard deviation for MD, as well as for FA. The mean (ρ) was used to assess any correlation between DTI measures including the measure ratios and GS. Systematic differences in DTI measures (including the ratios of the measures) between GS ≤ 7 (3 + 4) and GS ≥ 7 (4 + 3) risk group tumors were tested using a Mann–Whitney U-test. Receiver operating characteristic (ROC) curves were plotted and the areas under the ROC curves (AUC) were used as indices of performance for each measure in discriminating GS ≥ 7 (4 + 3) from GS ≤ 7 (3 + 4) risk group tumors. Statistical analyses were performed using SPSS software for Windows . P < 0.05 was considered statistically significant.Systematic differences in the DTI measures between the tumor and the noncancerous prostate tissue were tested using Wilcoxon signed-rank test. Spearman correlation coefficient (P < 0.05). On the other hand, FA based DTI measures from the tumors are higher than the ones from the noncancerous prostate tissue significantly (P < 0.05). These results verify the appropriate placement of the ROI for the noncancerous prostate tissue. Average contrast-to-noise ratio is calculated as 16.0 from MD maps and 3.1 from FA maps showing that MD maps provide better tumor contrast with respect to FA maps of DTI.DTI measures captured for all prostate tumor foci and for all noncancerous tissue are as seen in ρ = −0.73 to −0.60, P < 0.001). On the other hand, FA based DTI measures provide weak positive correlations with Gleason score. Among all DTI measures, MDmin has the highest correlation with GS (ρ = −0.73) while rMDmin is correlated with GS in a similar manner (ρ = −0.73).Box plots of MD and FA based DTI measures for all tumors stratified by GS groups are shown in max, FAmean, rFAmax, and rFAmean only (P < 0.05). The results of the ROC analyses of the measures in discriminating GS ≤ 7 (3 + 4) from GS ≥ 7 (4 + 3) risk group tumors are shown in min stands out in terms of its highest performance (AUC = 0.93); however MDmin shows almost the same performance (AUC = 0.92).Case summaries of the measures for GS ≤ 7 (3 + 4) and GS ≥ 7 (4 + 3) risk group tumors are as seen in Diffusion tensor imaging (DTI) provides mean diffusivity (MD) and fractional anisotropy (FA) that reflect molecular diffusion rate and diffusional anisotropy characteristics as sensitive measures of altered tissue structure. In this study, we investigate the utility of statistical measures  based on MD and FA and the ratios of these measures to exhibit the full potential of DTI in evaluating the aggressiveness of peripheral zone prostate cancer.The normal supporting stroma and capsule of the prostate consist of dense fibroelastic connective tissue in which prostate glands are dispersed. At the central zone of the prostate, these glands increase greatly in size as a normal part of the aging process. Conversely, the peripheral zone is the part of the prostate that most of the cancers are located. Normal peripheral zone prostate tissue is composed of three gland component volumes including stroma, epithelium, and lumen space. In the case of prostate cancer, the percentage volume of stromal tissue decreases because of the collection of small atypical glands. As GS and tumor grade increase, tumor cells either form cribriform, fused glands, and solid sheets or infiltrate as individual cells without forming luminal spaces . AlthougOur results from 38 patients with 42 identified prostate cancer tumors demonstrate that strong negative correlations are observable between GS and MD based tumor DTI measures in agreement with –16 and tIn discriminating GS ≤ 7 (3 + 4) from GS ≥ 7 (4 + 3) risk group tumors, our results show that systematic differences are present for all MD based measures as expected considering the strong negative correlations with GS. Among the FA based measures, the differences are present only for maximum tumor FA, mean tumor FA, ratio of maximum FA, and ratio of mean FA although these FA measures are correlated weakly with GS. MD based measures perform quite better than the FA based measures. The ratio of minimum MD from prostate tumor foci and noncancerous PZ prostate tissue stands out in terms of its highest discrimination performance. However, the minimum MD from prostate tumor foci alone provides almost the same remarkably high performance.Ratios of DTI measures tested in this study show similar performances of the measures alone in correlations with GS and in discriminating GS groups. While calculating the ratios, noncancerous peripheral zone prostate tissue is considered as the reference tissue. Distinguishable performance differences can be obtained when noncancerous transition zone prostate tissue, urinary bladder tissue, or urine is used as reference tissue. These tissues have been tested in DWI to improve the performance of ADC –21). On There are some limitations of the study. Histopathologic specimens of the tumors analyzed in this study were obtained from either prostatectomy or in-bore biopsy. The whole mount histologic specimens from prostatectomy reflect the tumor with highest accuracy. However, type of utilization may cause selection bias since radical prostatectomy material cannot be utilized from the patients with low-risk tumor on TRUS-bx included in active-surveillance regimens or the patients with high-risk disease and being candidates for hormone or radiation therapy. In-bore biopsy cases of this study prevent the possible selection bias. In in-bore biopsy, a direct match between the identified tumor and the corresponding histopathologic specimen can be obtained with good accuracy since the biopsy needle is targeted to the tumor under the guidance of MR device. b-values , and a voxel size of 3.6 mm3. More robust estimations for DTI measures that may lead to stronger correlations with GS especially for FA based measures can be obtained by using a larger number of diffusion encoding directions and b-values [b-values would result in an increase in the scanning time that may lead to severe motion artifact while making the imaging unfeasible for clinical practice. On the other hand, larger number of diffusion encoding directions may lead to lower values for FA measures [The diffusion tensor imaging protocol of this study employs 12 diffusion encoding directions, two differentb-values . Howevermeasures . The voxmeasures .In the current study, small ROIs have been used and T2-weighted images have been evaluated to identify the fibrous prostate tissue during the placement of the ROIs. Selection of the ROI size and the placement of the ROI require utmost attention for good repeatability and reliability for the DTI measures. Large differences in the measures can be experienced when the ROI size is larger or when the ROI is placed on fibrous tissue of the prostate gland.b = 0 s/mm2 during this study is equal to 58 on average. This value is far beyond the minimum SNR recommended  to obtain unbiased DTI measures [The signal-to-noise ratio measured from the DTI images acquired with measures . In addimeasures , 15.This work is focused on the clinical benefits of the DTI measures and of the ratio of these measures in assessment of peripheral zone prostate cancer aggressiveness. In the near future, we plan to assess the utility of noncancerous transition zone prostate tissue, urinary bladder tissue, and urine as reference tissue in ratio calculations and we plan to develop an automated method to obtain ROIs for the tumor foci and for the reference tissue. We also plan to extend our study to include the translational zone prostate cancer.In conclusion, the ratio of MD based diffusion tensor imaging measures can be used to determine the aggressiveness of peripheral zone prostate cancer; however, a similar diagnostic performance may be obtained by using the tumor MD measure alone.


Shc adaptor protein has demonstrated that aging-regulating genes and reactive oxygen species (ROS) are closely interconnected, since its absence modifies metabolic homeostasis by providing oxidative stress resistance and promoting longevity. p66Shc(−/−) mice are a unique opportunity to further comprehend the bidirectional relationship between redox homeostasis and the imbalance of mitochondrial biogenesis and dynamics during aging. This study shows that brain mitochondria of p66Shc(−/−) aged mice exhibit a reduced alteration of redox balance with a decrease in both ROS generation and its detoxification activity. We also demonstrate a strong link between reactive nitrogen species (RNS) and mitochondrial function, morphology, and biogenesis, where low levels of ONOO− formation present in aged p66Shc(−/−) mouse brain prevent protein nitration, delaying the loss of biological functions characteristic of the aging process. Sirt3 modulates age-associated mitochondrial biology and function via lysine deacetylation of target proteins, and we show that its regulation depends on its nitration status and is benefited by the improved NAD+/NADH ratio in aged p66Shc(−/−) brain mitochondria. Low levels of protein nitration and acetylation could cause the metabolic homeostasis maintenance observed during aging in this group, thus increasing its lifespan.Programmed and damage aging theories have traditionally been conceived as stand-alone schools of thought. However, the p66 Specific activity was calculated by subtracting the remaining activity in the presence of the NOS inhibitor 5 mM L-NMMA or 2 mM ethylene glycol tetraacetic acid . Me. MeShc mice show high levels of PGC-1α gene expression and increased mitochondrial content compared with their WT littermates  mouse brain mitochondria present enhanced ∆Ψ and display filament or tubular shape. This is consistent with the observed Mfn2 upregulation, a GTPase embedded in the mitochondria outer membrane directly involved in its fusion, and with the downregulation of Drp1 expression  mice throughout their lives. In accordance to these data, we show here that Sirt3 is sensitive to be nitrated and its activity is protected against age-related nitration in brains of aged p66Shc(−/−) mice  mice . These f2O2), p66Shc is a clear example of this strong link between oxidative stress and the genetic of aging, and it could be a potential target for anti-aging strategy. The modulation of p66Shc activity could be a candidate for therapeutic intervention for a longer lifespan or higher quality of life.In accordance to the free radical theory of aging, this process is triggered and sustained by the deleterious accumulative effect of oxidative damage caused by mitochondrial respiration-generated ROS. On the other hand, the genetic programmed theory asserts that aging and death are necessary parts of evolution, and the low variability in lifespan within species proves that aging is not only a wear-and-tear process –72. ThroShc inactivation in multiple processes related to aging in mouse brains. Through the modification in ROS/RNS production, which in turn regulates Sirt3 activity, the absence of p66Shc improves mitochondrial dynamics and biogenesis. Taken together, these findings can partially explain the observed delay in the aging of p66Shc(−/−) mouse brains. Moreover, the enhancement in mitochondrial homeostasis can be the underlying cause of the improved cognition and motor function observed in p66Shc KO mice during aging [In conclusion, results show the pivotal role of p66ng aging . More st


Pseudomonas species in environments with and without mercury selection. Plasmid fitness effects and stability varied extensively between host species and environments, as did the propensity for chromosomal capture of the Tn5042 mercury resistance transposon associated with loss of the plasmid. Whereas Pseudomonas fluorescens and Pseudomonas savastanoi stably maintained the plasmid in both environments, the plasmid was highly unstable in Pseudomonas aeruginosa and Pseudomonas putida, where plasmid-free genotypes with Tn5042 captured to the chromosome invaded to higher frequency under mercury selection. These data confirm that plasmid stability is dependent upon the specific genetic interaction of the plasmid and host chromosome rather than being a property of plasmids alone, and moreover imply that MGE dynamics in diverse natural communities are likely to be complex and driven by a subset of species capable of stably maintaining plasmids that would then act as hubs of HGT.Mobile genetic elements (MGE) such as plasmids and transposons mobilise genes within and between species, playing a crucial role in bacterial evolution via horizontal gene transfer (HGT). Currently, we lack data on variation in MGE dynamics across bacterial host species. We tracked the dynamics of a large conjugative plasmid, pQBR103, and its Tn5042 mercury resistance transposon, in five diverse  Bacteria share genes via mobile genetic elements but the dynamics of these vary extensively between species. Our experimental system consisted of a conjugative plasmid, pQBR103, conferring mercury resistance via a Pseudomonas species  isolated from a range of environments: P. fluorescens SBW25 was isolated from the leaf surface of the sugar beet plant  is a denitrifying soil isolate   marker using the mini-Tn7 transposon system  P. fluorescens SBW25 stocks that were carrying pQBR103 plasmid, with the plasmid-free GmR strains for 48 h and spreading on KB agar plates containing 10 μg mL−1 gentamicin and 20 μM of mercury (II) chloride to select for transconjugant colonies  chloride concentrations from 0 to 60 μΜ. Relative fitness was measured by mixing differentially labelled test  and reference  in ∼1:1 ratio, diluted 100-fold in KB microcosms containing the relevant mercury concentration and incubated at 28°C for 48 h. Samples were plated on KB agar plates at the beginning and end of the competition and replica plated onto KB agar plates supplemented with selective concentration of gentamicin to estimate the density of plasmid bearers. The relative fitness was calculated as the selection rate (r) , after which time 1% of each population was transferred to grow for 24 h in fresh KB microcosms containing 50 μΜ of mercury (II) chloride at the same temperature and shaking conditions. This step was necessary to ensure high starting frequencies of plasmid carriage across all the tested bacterial strains due to the high segregation rate of the plasmid in some host strains .Prior to the evolution experiment 12 individual colonies of each et al.mer operon on Tn5042 transposon  and the other to origin of replication of the plasmid (oriV) . Detection limits were estimated by Poisson calculations.For the evolution experiment, six clonal populations of each bacterial strain were grown in absence of mercury and six populations were grown in the presence of mercury selection (50 μΜ of mercury (II) chloride) in KB microcosms at 28°C with shaking (180 rpm/min). Every 48 h 1% of each population was transferred into a fresh microcosm for 60 transfers [∼400 generations]. The density of each bacterial population was monitored every four transfers by plating a sample onto KB agar plates incubated at 28°C for 48 h. Each plate was then replica plated onto KB agar plates supplemented with 100 μΜ of mercury (II) chloride to assess the frequency of mercury resistance. Subsequently, 24 bacterial colonies were randomly selected from each mercury replica plate. The prevalence of the mercury resistance transposon, Tn5042, and the plasmid, pQBR103, was estimated at transfers 2, 4 and 8 and then at every 12 transfers by PCR screening of the 24 bacterial colonies randomly selected from each clonal population. The PCR screening was designed as previously described  chloride). Plasmid population dynamics in the P. stutzeri parallel evolving populations were further investigated by comparing plasmid prevalence across mercury conditions using a one way-ANOVA. Plasmid prevalence was estimated as the area under the curve using the function auc of the package ‘flux’ , ANOVA F1,32 = 72.829, P = 9.425e−10), the fitness of the other plasmid bearing Pseudomonas species increased rapidly at low concentrations of mercury (>/ = 7.5 μM mercury (II) chloride) . Model comparison revealed that the fitness response to mercury concentration was non-linear in 4/5 of the species . Furthermore, minimum inhibitory concentration assays showed that species varied both in their inherent susceptibility to mercury  and in the level of mercury resistance conferred by pQBR103 . These data demonstrate extensive variation in the fitness effect of plasmid acquisition across the Pseudomonas phylogeny, suggesting that even in relatively closely related bacteria , plasmids and their accessory genes can have markedly different fitness effects.We first quantified the fitness effect of plasmid carriage on bacterial hosts using competition experiments across a wide range of mercury environments, ranging from no mercury where the plasmid confers no benefit (0 μM mercury (II) chloride) to high levels of mercury contamination where the plasmid and its mercury transposon are essential (60 μM mercury (II) chloride). Although, in general, the plasmid was costly to host species in the absence of mercury, the magnitude of the cost and the form of the fitness response with increasing mercury concentration varied between species . As expected, mercury selection promoted the maintenance of mercury resistance in all host species. Without mercury selection, the maintenance of mercury resistance was highly dependent on host species . In P. aeruginosa, resistance dynamics varied across replicate populations, with final frequencies ranging from 4% to 80% of the population evolving in mercury-free environment (>95% probability of detecting HgR if present at frequencies of ≥3.2%).To examine the consequences of these fitness effects on long-term population dynamics, we tracked mercury resistance in populations evolved for ∼400 generations either with or without mercury selection. Mercury resistance was assessed by replica plating populations every four transfers onto mercury selective media. At the end of the experiment, we found that the level of mercury resistance varied between ies Fig. . In the et al.Pseudomonas host species . In contrast, we observed rapid, complete loss of the plasmid in P. putida and P. aeruginosa regardless of mercury selection (>95% probability of detecting pQBR103 if present at frequencies ≥2.1%), and where mercury resistance was observed in these hosts this resulted from chromosomal capture of the resistance genes   to a clear mutualist (enhancing the relative fitness of its host by 71%), similar to previous work , but measurements of fitness can only partly explain the long-term dynamics. For example, long-term plasmid population dynamics in P. fluorescens and P. putida were widely divergent despite the fact that costs-of-carriage were similar. Rates of segregation and/or conjugation, which are known to vary between species . If maintenance and spread of plasmids is governed more by species presence than by selection for plasmid-borne genes, this has important implications for the control of resistance elements: without an understanding of the species involved, attempts to limit the spread of resistance by limiting antibiotic use (for example) may prove to be unsuccessful.Our work has shown that long-term plasmid-host dynamics vary with environmental selection and host genotype. Laboratory experiments such as these are revealing a complex and contingent partnership but are necessarily simple, stripping away many of the details of the natural environment. Natural environments are likely to be spatially structured and heterogeneous, which could act to both promote and impede HGT between species; spatially structured environments can potentially impede encounters between cells, reducing opportunities for HGT, whereas, in such environments, biofilm growth predominates and environmental heterogeneity can promote species coexistence, which could increase opportunities for interspecific HGT (Heuer and Smalla FEMSEC online.Supplementary data are available at Supplementary materialFEMSEC online.Supplementary data are available at Click here for additional data file.


The loss of vision following Boston Keratoprosthesis (BKPro) surgery due to glaucoma occurs at a high frequency as diagnosis and management of glaucoma after this procedure pose challenges.To compare visual outcomes in patients undergoing Boston Keratoprosthesis surgery with and without prior or concurrent glaucoma surgery.This is a retrospective, observational cohort study of patients who underwent Boston Type I Keratoprosthesis surgery. 19 eyes of 18 patients who had undergone BKPro and met the inclusion criteria were identified. Twelve eyes received BKPro with prior or concurrent glaucoma surgery (Group 1), and seven eyes were identified undergoing BKPro surgery without prior or concurrent glaucoma surgery (Group 2).Main outcome included best corrected visual acuity at each follow up.In Group 1, mean best corrected visual acuity (BCVA) within a year of BKPro surgery was 20/100 (range 20/40 to Count Fingers (CF); n = 12) and mean BCVA at 1 year from BKPro surgery was 20/115 . 7 out of 12 patients retained or had improved BCVA at 1 year follow up after BKPro implantation, and 5 out of 12 patients had mild BCVA worsening. In Group 2, the mean BCVA within a year of BKPro surgery was 20/140 (ranging from 20/25 to hand motion vision (HM); n = 7) and mean BCVA at 1 year from BKPro surgery was Count Fingers (range 20/60 to Light Perception (LP); n = 6). 4 out of 6 patients lost significant vision at one year after BKPro.BKPro patients with early glaucoma surgical intervention retained vision significantly better compared to patients with late or no intervention. Our preliminary findings support the recommendation for concurrent or pre-emptive glaucoma surgical intervention in patients undergoing BKPro implantation. The restoration of a clear visual axis in patients who suffer from corneal blindness can present challenges in those who are also poor candidates for traditional corneal transplantation techniques. These patients often have failed multiple prior penetrating keratoplasties, have ocular surface scarring due to trauma or autoimmune disease, and/or have limbal stem cell deficiencies. The Boston Keratoprosthesis (BKPro) is a prosthetic corneal device that offers an option to restore vision in this cohort of patients. The initial technique was FDA approved for use in 1992 and has evolved to reduce many of the initial shortcomings. This has resulted in more frequent use and greater success in restoring vision in those who are poor candidates for penetrating keratoplasty.The design of the BKPro consists of 3 main components. There is a corneal graft, which is enveloped between a central optical polymethyl methacrylate (PMMA) button and a posterior plate. This design allows a fixed clear central visual axis with peripheral corneal graft tectonic support to suture to the globe. Although surgeon experience has resulted in improved outcomes and more successful adoption of this technique, there remain several frequently encountered post-operative challenges. These challenges include the development of retroprosthetic membranes , a high Patients undergoing traditional penetrating keratoplasty are already at increased risk of developing glaucoma due to angle closure or as a result of steroid induced elevation of intraocular pressure. Traditionally, glaucoma after penetrating keratoplasty can be identified through measurement of intraocular pressure and measurement of visual field and treated with traditional methods such as topical medications or glaucoma filtering surgery. Patients undergoing prosthetic corneal transplantation with BKPro may be at the same or greater risk of developing glaucomatous disease after surgery –5, howevDue to this imprecise measurement of IOP, many patients who do develop glaucoma after BKPro suffer glaucomatous vision loss as a result of delayed diagnosis. Even when elevated IOP is identified postoperatively, the success of topical medications in lowering IOP is challenging to assess and other traditional methods of glaucoma monitoring such as visual field testing and optic nerve imaging are not reliable. Furthermore, should glaucoma surgery be indicated after BKPro, the anatomical restrictions from limited anterior chamber visualization make it preferable to adopt a pars plana implantation of a glaucoma filtering implant with pars plana vitrectomy. Given the frequency of vision loss after BKPro, glaucoma surgery may be of benefit when planned concurrently with BKPro as a measure to prevent vision loss from uncontrolled IOP. In this study, we aim to investigate the relationship between vision retention and early versus late surgical glaucoma intervention in patients with type 1 Bkpro.This is a retrospective, observational cohort study of all patients who underwent Boston Type I Keratoprosthesis surgery between the dates of January 1, 2006 through December 31, 2016 at the University of California, Irvine Ophthalmology Clinic  and Gavin Herbert Eye Institute . All Boston type I Keratoprothesis were performed by a single surgeon and identified using CPT codes for Keratoprosthesis surgery. Institutional Review Board Approval at UC Irvine was obtained for this review, and a waiver for patient consent was granted as this was a retrospective review. 34 eyes of 22 patients were initially identified who had undergone BKPro. A minimum of six months of follow-up was required for inclusion in the study. Eyes that required repeated BKPro, retinal detachment repair, or enucleation during the follow-up period were excluded from the study. Additionally, eyes that had severe retroprosthetic membrane formation, stromal melt, or infectious keratitis that required explanation of BKPro or placement of a new BKPro during the follow-up period were excluded. After applying inclusion and exclusion criteria, 19 eyes of 18 patients were included in this review, and were separated into two groups. Group 1 underwent glaucoma surgery at the time of BKPro or within 3 months of BKPro, and the other group either did not undergo glaucoma surgery or underwent surgery later than 3 months postoperatively. Primary outcome was BCVA at 6 (± 1) months, 12 (± 2) months, 24 (± 2) months, and 36 (± 2) months post-BKPro. BCVA following visual acuity stabilization within 12 months post-BKPro was also measured. Visual Acuity was initially measured with the Snellen chart then converted to a continuous Logarithm of the Minimal Angle of Resolution (Log MAR) units for quantitative analysis. For non-numerical values, the conversions are as follows: Count Fingers = 1.7, Hand Motion = 2.0, Light Perception = 2.3, and No Light Perception (NLP) = 3.0. Each patient’s age, gender, previous surgeries, pre-operative number of glaucoma medications and pre-operative IOP, as measured with applanation or indentation tonometry, were recorded as part of baseline characteristics . OriginaReliable visual fields, optic disc imaging, retinal nerve fiber layer analysis, or objective post-op IOP measurements were unavailable for each patient at consistent time points post-operatively to be meaningfully compared between the groups, and thus were not reported.t-test assuming unequal variance, and comparison of mean visual acuity in LogMAR was achieved with the Mann-Whitney U nonparametric test. Differences among categorical values between the groups were assessed using Fisher’s exact test (2-tailed). Statistical significance was defined as p < 0.05 for all tests.Continuous variables were expressed and tabulated as mean values with corresponding standard deviations. Standard error of mean was represented in graphical form. Categorical values were expressed as discrete integers, frequencies, and/or percentages. Comparison of baseline characteristic means between the two groups was achieved with independent Student’s Nineteen eyes of 18 patients who underwent Boston Type I Keratoprosthesis implantation meeting the inclusion criteria were identified. Of these 19 eyes in the study, there were no documented cases of endophthalmitis or vitritis during the follow-up period. Twelve eyes of 11 patients received prior glaucoma surgery (n = 8) or glaucoma drainage implants concurrently (n = 4) with BKPro (Group 1). Of the remaining seven eyes in seven patients, six had never received any glaucoma surgery, and one patient received glaucoma surgery 2.5 year after BKPro (Group 2). Of note, two patients in Group 2 received cyclophotocoagulation six months and two years after BKPro, respectively. Preoperative best corrected visual acuity (BCVA) ranged from 20/400 to HM for both groups (with the median being CF in both), with an average LogMAR of 1.76 ± 0.26 for Group 1 and 1.81 ± 0.27 for Group 2 (p = 0.65). All patients had retained or improved BCVA within 3 months after BKPro (median 20/150 in Group 1 and 20/400 in group 2). Mean BCVA before and after BKPro at several time points are displayed in Loss of vision, as defined by the difference in LogMAR between best corrected vision after stabilization within 12 months and best corrected vision at 12, 24, and 36 months post-BKPro are shown in Breakdowns of BCVA by each individual eye within 1 year post-BKPro and then at 1 year are shown in The use of the BKPro to restore vision in corneal blindness has been a significant milestone in therapy to offer to patients who previously had severely limited options for vision restoration. Although initial adoption of this procedure was slow, it has been more frequently offered in the last decade as surgeons have encountered better outcomes. Much of this success in improving outcomes using the BKPro procedure can be attributed to improved management of the most common post-operative issues –9. RetroThis retrospective review demonstrates better visual acuity retention one-year post operatively in patients who undergo glaucoma surgical intervention concurrently or close to the time of BKPro surgery. What is also of equal importance is the incidence of significant vision loss in the group of patients undergoing BKPro without close or concurrent glaucoma surgery. Although there are limitations in the outcome measures and it is difficult to accurately ascertain all causes of BCVA loss following BKPro, these results may suggest a trend of worsening vision loss over time when glaucoma is not prophylactically addressed at the time of BKPro surgery.Surgeons have reached a consensus in measures to reduce the impact of retroprosthetic membranes and endophthalmitis after BKPro. Perhaps anticipating the need for glaucoma surgery at the time of BKPro can reduce the impact of vision loss from glaucoma post operatively. The way in which glaucoma surgery can be offered with BKPro requires careful consideration. Given the limitations in visualizing the peripheral anterior chamber after BKPro and the higher rates of postoperative infection, standard approaches to external filtration surgery are difficult. A trabeculectomy bleb may increase the lifetime risk of infection in an eye already prone to endophthalmitis, and placement of a tube into the anterior chamber or sulcus is precluded without the necessary visualization. In the past, authors have employed cyclodestructive procedures to be used in conjunction with the BKPro surgery with some success . HoweverEven in patients who have successful coordination of glaucoma surgery at the time of BKPro with stable BCVA over time, glaucoma has the potential to worsen and present as a later complication to BCVA. Digital applanation, although a crude method even with expert examiners, still offers some ability to gauge IOP when compared with the patient’s contralateral eye where Goldmann applanation may be possible. In patients where BCVA and cooperation allows serial visual field testing and optic nerve structural analysis, these tools may allow for close monitoring and escalation of treatment as needed. In patients where these tools are not possible, serial examination of the optic nerve head and comparison with baseline optic nerve photos may be a useful tool. If IOP control is in doubt, escalation of treatment with topical glaucoma medications should be strongly considered.There are numerous limitations to this study. Firstly, there is no way to directly identify glaucoma as the main cause of vision loss in this group of subjects. While vision loss may be secondary to numerous contributing causes, our study does correlate early surgical intervention with better retention of vision. Secondly, this is a retrospective study with all the inherent limitations therein. Also, the number of subjects is limited, particularly in the late-intervention group. With a small sample size, adjustments based on age, gender, race, baseline IOP, or comorbidities would not yield meaningful conclusions. We also acknowledge that since visualization of the retina, optic nerve head, and measurement of IOP are challenging in these patients, comorbidities and complications such as retinal pathologies, suprachoroidal hemorrhage, and hypotony maculopathy cannot be consistently elicited and reliably gathered from all patient’s charts. These factors may confound visual acuity post-operatively. Lastly, selection bias may exist for patients with worse glaucomatous disease at baseline to undergo concurrent intervention. If such bias confounds our findings, however, they would likely further strengthen the relationship between early intervention and improved visual acuity outcomes.In summary, we observed increased vision retention at 12 months post-op for patients who underwent early or concurrent glaucoma surgery with BKPro compared to patients who received late or no glaucoma surgery. Our findings suggest that prophylactic glaucoma surgery may be beneficial for patients undergoing K-pro implantation. Larger-scale prospective studies are needed to confirm the strength of this relationship and further characterize the complication profiles versus clinical benefit of concurrent glaucoma surgery with K-pro.S1 Table(XLSX)Click here for additional data file.


Food intake and body weight was monitored daily during 3 weeks. Glucose homeostasis was assessed from glucose and insulin tolerance tests. In obese WT and MOR−/− mice, DJB induced a rapid and sustained weight loss partly independent of food intake, and a rapid improvement in glycaemic parameters. Weight loss was a major determinant of the improvements observed. In lean WT and MOR−/− mice, DJB had no effect on weight loss but significantly enhanced glucose tolerance. We found that MORs are not essential in the metabolic beneficial effects of DJB, suggesting that protein sensing in the distal gut is not a link in the metabolic benefits of gastric surgery.Roux-en-Y gastric bypass (RYGB) induces remission or substantial improvement of type 2 diabetes mellitus (T2D) but underlying mechanisms are still unclear. The beneficial effects of dietary proteins on energy and glucose homeostasis are mediated by the antagonist effects of peptides toward mu-opioid receptors (MORs), which are highly expressed in the distal gut. We hypothesized that the beneficial effects of RYGB could depend at least in part on the interaction of peptides from food with intestinal MORs. Duodenal-jejunal bypass (DJB) was performed in obese and lean wild-type (WT) or MOR deficient (MOR Bariatric surgeries have emerged as the most effective and durable therapies to treat obesity and one of its comorbidities, type 2 diabetes mellitus (T2DM)124A particularity of RYGB is that the gut anatomy is changed, which results in a modification in nutrients routing. It is well known that macronutrients act as signalling molecules and, in particular, proteins are known to induce beneficial effects on health. Protein-enriched diets have proven their efficacy in promoting satiety791012−/−) mice to the satiety effects induced by protein-enriched diet1516Recently, we demonstrated that mu-opioid receptors (MORs) are essential in mediating the satiety effects of dietary proteins−/− mice.In this study, we assessed whether MORs are involved in the short-term metabolic effects of RYGB, either in a context of obesity or of leanness. Since we were primarily interested in the role of gut remodelling in the beneficial effects of RYGB, we performed a duodenal-jejunal bypass (DJB) surgery, which is a simplified RYGB that does not imply any gastric volume restriction , in bothAll the experiments involved in this study were carried out in accordance with the principles and guidelines established by the European Convention for the Protection of Laboratory Animals and approved by our regional animal care committee .−/−) were generated in our facilities from two couples of mice obtained from The Jackson Laboratory (Oprm1tm1Kff/J)Male C57Bl/6 J wild-type mice (WT) were purchased from Charles River Laboratories at 4 weeks of age. Mu-opioid receptors knock-out mice (MOR−/− mice were given ad libitum a high-fat/high-sucrose (HF-HS) diet during 20 weeks prior to surgery. HF-HS diet, consisting of 36.1% fat (butter and soybean oil (14:1)), 35% carbohydrates  and 19.8% proteins (casein from bovine milk), was produced by the Unité de Préparation des Aliments Expérimentaux . For studies in lean animals, WT and MOR−/− mice were maintained on standard diet  and surgery was performed at 14-weeks old. All the animals were maintained on their respective diet after surgery.In order to induce obesity, 4-weeks old WT and MOR®) was applied on mice eyes to prevent drying and the abdomen was shaved and disinfected. During the entire operation, organs exposed to air were kept moist by application of NaCl 0.9%.Food and water were not restricted before surgery. Anaesthesia was induced and maintained throughout the operation with Isoflurane and air/oxygen. During anaesthesia, mice were placed on a temperature-controlled heating pad to avoid hypothermia. Ophthalmic ointment  underwent the same duration of anaesthesia as DJB-treated mice (1h15) and the abdominal wall and skin were closed using running and interrupted suture respectively (Ethicon 6-0 nylon). The sham-PF mice were pair-fed to match the daily food intake of the DJB mice.ketoprofen (5 mg/kg) was administered by an intraperitoneal injection before the abdominal wall closure and 1.5 mL of NaCl 0.9% was injected subcutaneously at the end of the surgery. Postoperative analgesia consisted in a daily injection of ketoprofen  for 4 days after surgery. Mice were housed in individual cages and put on heating pad set at 37 °C during 5 days. After the surgery, mice were given ad libitum access to water but were deprived of food during 24 h. In the postoperative day 1, mice were given approximately 2 mL of liquid diet (Ensure). From the second postoperative day, mice were fed ad libitum with the appropriate food (HF-HS or standard diet), except for sham-PF mice.Body weight and food intake was monitored daily during 3 weeks. HF-HS diet was changed 3 times a week for ad libitum fed mice (sham-operated and DJB-treated mice) and daily for sham-PF mice.Glucose tolerance test (GTT) and insulin tolerance test (ITT) were carried out 2 and 3 weeks after the surgery, respectively. Animals were fasted for 16 (GTT) or 6 hrs (ITT) and then received an intraperitoneal injection of glucose (1 g/kg) or insulin . Blood glucose was monitored for 120 (GTT) or 90 min (ITT) using a glucometer  From samples collected from the tip of the tail vein. Insulinemia was quantified using an ELISA kit (Mercodia).The results were expressed as mean ± SEM. Unpaired Student’s t test was used for two-group comparisons. One-way ANOVA followed by Tukey’s post-hoc test was used for three-group comparisons. p < 0.05 was considered significant.th day post-surgery, WT DJB mice stabilized their food intake and reached the same level as that of WT sham mice. These results indicated that DJB did not induce a long lasting decrease in food intake. However, DJB in WT obese mice led to a significant and sustained weight loss compared to sham surgery. Indeed, WT DJB mice showed a steeply weight loss for the first 15 days  diet. Food intake after DJB was transiently reduced from postoperative day 3 to day 8 compared with WT sham group . Beyond Then we evaluated the effects of DJB on glycaemic control. Analysis of glucose tolerance test data revealed that DJB induced a significant improvement in glucose tolerance associated with a decrease in basal glycaemia  and a 3 −/− obese mice. As in WT mice, DJB in MOR−/− obese mice led to an initial hypophagia from postsurgical day 3 to day 6 and then to a stabilization in their food intake at the same level as MOR−/− sham mice  and regained weight from the 9th post-surgical day until the end of the experiment . Together, these data indicated that DJB performed in lean WT and MOR−/− mice induces beneficial effects on glucose tolerance that are specific of surgery and independent of weight loss.Insulin tolerance data demonstrated that neither WT DJB mice nor MORPrevotella copri28Akkermansia muciniphilaAkkermansia muciniphila is increased after gastric bypass in morbidly obese patients31This paper addresses the question of the effects of DJB on glucose and energy homeostasis in obese and diabetic mice fed a high-calorie diet. We highlight that DJB in the mouse is a suitable model to analyse the metabolic consequences of the rearrangement of the intestinal tract produced by RYGB surgery. Indeed, obese mice undergoing DJB surgery exhibited a 30% weight loss and significant improvements in glucose tolerance and insulin sensitivity as observed after RYGB in humans. However, in this mouse model of DJB, the weight loss occurred within 2 weeks after the surgery, while in humans the same weight loss was observed only 6–12 months after RYGB. This substantial and early weight loss is nonetheless consistent with the findings of others that performed either RYGB212223212426per se to confer the improvements in glucose metabolism, confirming the studies with mice that were body-weight matched with RYGB mice2434Regarding the consequences of DJB on glucose homeostasis in obese and diabetic mice, our results suggest that the significant improvement in glucose tolerance was directly correlated to body weight. Moreover, insulin tolerance improved in proportion to weight loss. Adiposity was not assessed here. However, the skeletal muscle greatly contributes to glucose control. This warrants why body weight loss monitoring was preferred to adiposity monitoring in this study. The results obtained highlight that the early and substantial weight loss occurring after DJB might be sufficient −/− mice to address this question. We first noted that these mice displayed a lower body weight than WT mice when they were fed a HF-HS diet. These results are consistent with data from the literature that revealed that MOR−/− mice consuming a high-calorie diet gain less body weight than WT mice because of a lesser fat storage36−/− obese mice, as in WT obese mice. Moreover, the positive correlations between glucose homeostasis and weight and between insulin sensitivity and weight loss induced by DJB were observed in MOR−/− mice. This suggests that MORs are not implicated in the glycaemic parameters improvements in DJB. However, the possible role of MORs might be hidden by the benefits deriving from the dramatic weight loss in obese mice.Using this mouse DJB model, we studied the possible role of dietary protein in the beneficial effect of DJB. Since MORs are a key mechanistic link in the favourable effects induced by protein-enriched diets−/− and WT mice. However, it is noteworthy that the effect of DJB on body weight in lean MOR−/− mice was more pronounced than in lean WT mice  DJB in obese mice promotes a sustained weight loss, which cannot be explained by the only decrease in food intake; (2) DJB improves glucose control in obese mice, which is mainly dependent on the decrease in body weight; (3) MORs and by extension dietary protein sensing are likely not involved in the beneficial outcomes induced by DJB. Therefore, this work shows that DJB surgery in mice is a suitable model to decipher the mechanisms underlying the rapid T2D improvement after RYGB, since DJB is less difficult to realize than RYGB and promotes the same beneficial effects on body weight and glucose homeostasis. Since benefits induced by bariatric surgery seem to be the result of a combination of several complex mechanisms, studies from various mouse models  combined with pair-feeding/weight-matching approaches should be useful to improve our knowledge in the mechanisms underlying gastric bypass surgeries.How to cite this article: Barataud, A. et al. Absence of Role of Dietary Protein Sensing in the Metabolic Benefits of Duodenal-Jejunal Bypass in the Mouse. Sci. Rep.7, 44856; doi: 10.1038/srep44856 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The BRCA2 gene product plays an important role in DNA double strand break repair. Therefore, we asked whether radiation sensitivity of pancreatic cancers developing in individuals with germline BRCA2 mutations can be enhanced by agents that inhibit poly (ADP-ribose) polymerase (PARP).We compared the sensitivity of two patient-derived pancreatic cancer xenografts, expressing a truncated or wild type BRCA 2, to ionizing radiation alone or in combination with olaparib (AZD-2281). Animals were treated with either a single dose of 12Gy, 7 days of olaparib or 7 days of olaparib followed by a single dose of 12Gy. Response was assessed by tumour growth delay and the activation of damage response pathways.The BRCA2 mutated and wild type tumours showed similar radiation sensitivity, and treatment with olaparib did not further sensitize either model when compared to IR alone.While PARP inhibition has been shown to be effective in BRCA-mutated breast and ovarian cancers, it is less well established in pancreatic cancer patients. Our results show no radiosensitization in a germline BRCA 2 mutant and suggest that combining PARP inhibition and IR may not be beneficial in BRCA 2 related pancreatic tumors. Radiation therapy (RT) plays an important role in the treatment of locally advanced pancreatic cancers, but its effect is limited by the sensitivity of adjacent normal tissues, and the innate radioresistance of these cancers PMID:24462333). Exposure to ionizing radiation (IR) induces a variety of DNA lesions, of which DNA double strand breaks (DSB) are the most lethal, and, if left unrepaired, lead to genomic instability or cell death. The repair of DSB can be accomplished by two distinct DNA damage repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR) , 2. Homo4462333. Non-homologous end joining, which is active during all cell cycle phases, is the main DSB-repair mechanism activated in response to exposure to IR. This pathway catalysis a simple rejoining of two DNA DSB ends without guidance from a template and, as a result, is an error-prone process.PARP1 is a member of the poly-ADP-ribose polymerase family, a group of enzymes that has been shown to be involved in many processes including DNA repair and cell death , 9. PARPBecause of the essential role of PARP in DSB recognition and repair, PARP inhibitors might sensitize HR defective tumors following exogenous DSBs induced during treatment with IR, resulting in DNA DSB accumulation and cell death.Consistent with previous observations in a number of solid cancers –15, we oSubcutaneous tumors of two primary xenografts, designated as Ontario Cancer Institute Pancreas (OCIP) 23 and 28 were established from pancreatectomy samples superfluous to patient diagnosis using a protocol approved by the University Health Network Research Ethics Board as described previously , 17. Infhttp://www.ncbi.nlm.nih.gov/clinvar/RCV000044800) has a clinically-relevant, deleterious germline mutation in BRCA2 that has been described previously [Patient OCIP28  were treated for 7 days with 150mg/kg olaparib by oral gavage (po), a single dose of 12Gy or both according to the schedule shown in Olaparib was obtained from UHN Shanghai R&D . Treatments were started when tumor volume reached approximately 150mm2×length×0.5. Mean tumor growth delay (TGD) is defined as the difference in time (days) for treated animals to develop tumors that are 3.5 times or 5 times the initial tumor volume for OCIP23 and OCIP28, respectively, compared with animals treated with vehicle control. TGD was calculated by subtracting mean time to change in tumor volume for control animals from mean time to change in tumor volume of the various treatment groups.Subcutaneous tumors were measured using callipers, and volume calculated according to the formula widthTumors were excised, fixed and paraffin embedded. Paraffin tissue sections of 4μm thickness were cut, dried and dewaxed. KU70 staining was performed according to the manufacture’s guidelines using BenchMark XT-an automated slide strainer  with standard antigen retrieval  followed by incubation with the anti- KU70 antibody (1:400)  for 60 minutes. The Ventana Ultraview Universal DAB Detection Kit (#760–500) was used for secondary antibody incubations and slides were counterstained with Bill modified hematoxylin.For γH2AX staining, endogenous peroxidase was blocked and slides were incubated in γH2AX mouse monoclonal antibody . Biotinylated anti-mouse IgG incubations were carried out followed by streptavidin biotin detection system  for 30 minutes each. Immunoreactivities were revealed by incubation in Nova Red substrate  for 5 minutes and slides were counterstained in Gill modified hematoxylin.DNAPK and XRCC4 staining was performed according to the manufacture’s guidelines using BenchMark XT-an automated slide strainer with standard antigen retrival . Slides were incubated in DNAPK antibody  for 32 minutes or XRCC4 antibody  for 60 minutes. Ventana Ultraview Universal DAB Detection Kit (#760–500) was utilized for detection and slides were counterstained with Bill modified hematoxylin.Sections were scanned at 20× resolution using an Aperio Scanscope XT scanner . Images were analyzed using the Aperio ImageScope software ver. 11.1.2.752, positive pixel count algorithm (PPC). Necrotic areas were excluded from the analysis.Tumor growth data were analyzed using mixed effect modeling which accounts for correlations among the measurements of the same model. To determine whether the effect of treatment on growth rate differed between OCIP23 and OCIP28, another mixed effects model that included interactions between treatment, xenograft model and time was used. The significance of interaction terms was determined using Wald tests. The survival percentages for the in vivo data were calculated using the Kaplan-Meier technique and the curves were tested for significance using the log-rank test. The Kruskal-Wallis test was used to determine if treatment was associated with the percent of DNAPK, Ku70, XRCC4 and γH2AX. The Mann-Whitney test was employed to make pairwise comparisons between the four treatment groups.To test the radiosensitivity of the BRCA2 mutant (mt) and BRCA wt xenografts, tumor-bearing animals were treated with a single dose of 12Gy. Significant growth inhibition was seen in both models  compared with untreated control mice , and surTo investigate the potential efficacy of the combination of olaparib and IR, tumor bearing mice were treated with 150mg/kg olaparib, 12Gy of IR or the combination of olaparib and IR according to the schedule shown in No loss in body weight was observed in response to treatment .Treatment with olaparib alone did not affect tumor growth in either xenograft model  when compared to untreated controls . While IWe have previously demonstrated that the BRCA2 mt OCIP28 shows aberrant Rad51 foci . TherefoKu70 expression in OCIP23 was not impacted by treatment with either olaparib (p = 0.34) or IR (p = 0.081) when compared to untreated control tumors. However, the combination treatment significantly reduced Ku70 expression (p = 0.02) compared to irradiation alone Figs  and 4B. Previous studies have suggested that treatment with PARP inhibitors in combination with DNA damaging agents such as cisplatin or IR can result in the accumulation of DSB and γH2AX foci , 20. In We have recently shown that germline mutations of BRCA1 and BRCA2 sensitize pancreatic cancers to treatment with cisplatin and gemcitabine . Since tPARP is an important protein in DNA repair pathways especially in BER where PARP is involved in repair of SSBs. While PARP inhibition is functionally silent in HR proficient cells, PARP inhibition leads to the accumulation of SSBs and the development of DSBs in cells deficient in HR , 20. A nIn addition to its function in BER, PARP has also been shown to be involved in the regulation of DSB repair pathways , 18, 27.in vitro but failed to reduce tumor growth in vivo [In order to investigate whether the radiosensitivity of HR deficient tumors can be increased with the use of PARP inhibitors, we further investigated the efficacy of olaparib in combination with IR. We observed rapid resolution of DNA DSB breaks in the HR deficient patient-derived xenograft model in response to PARP inhibition. This increase in DNA damage repair may be attributed to the dysregulation of NHEJ and activation of DNAPK, resulting in low levels of persistent DSBs and rapid dephosphorylation of DNAPK that was not observed in the HR proficient xenograft model. In contrast to results shown in prostate cancer , no radi in vivo .Early observations suggest that pancreatic tumors show unusually high levels of chromothriptic events resulting in structural rearrangements, switches in copy number state and retention of heterozygosity –30. ExpoWhile recent studies have increased our knowledge of the regulation of DNA DSB repair mechanisms and PARP function, further studies are needed to fully understand the complex interactions between different pathways and the survival strategies that are “hitchhiked” by pancreatic cancer cells to evade persistent DNA damage and cell death.


Recent work suggests that the cytotoxic T lymphocyte (CTL) response to infection is an important defense mechanism in response to ZKV. Here, we develop the rationale and strategy for a new approach to developing cytotoxic T lymphocyte (CTL) vaccines for ZKV flavivirus infection. The proposed approach is based on recent studies using a protein structure computer model for HIV epitope selection designed to select epitopes for CTL attack optimized for viruses that exhibit antigenic drift. Because naturally processed and presented human ZKV T cell epitopes have not yet been described, we identified predicted class I peptide sequences on ZKV matching previously identified DNV (Dengue) class I epitopes and by using a Major Histocompatibility Complex (MHC) binding prediction tool. A subset of those met the criteria for optimal CD8+ attack based on physical chemistry parameters determined by analysis of the ZKV protein structure encoded in open source Protein Data File (PDB) format files. We also identified candidate ZKV epitopes predicted to bind promiscuously to multiple HLA class II molecules that could provide help to the CTL responses. This work suggests that a CTL vaccine for ZKV may be possible even if ZKV exhibits significant antigenic drift. We have previously described a microsphere-based CTL vaccine platform capable of eliciting an immune response for class I epitopes in mice and are currently working toward  The CDC estimates at least 4,100 cases in the United States and up to 29,000 cases in Puerto Rico. Those cases in Puerto Rico include 672 pregnant women , the pub1.1ZKV is a small enveloped plus strand RNA virus belonging to the genus Flavivirus, which includes many human pathogenic viruses, such as Dengue virus (DNV), yellow fever virus (YFV), West Nile Virus (WNV), and hepatitis C virus (HCV). ZKV has a 10.8 kb RNA genome, containing a single open reading frame flanked by a 5′-UTR (106 nt long) and a 3′-UTR (428 nt long). The open reading frame encodes a polyprotein precursor, which is processed into three structural proteins  and seven non-structural proteins . The viral E protein is the major surface glycoprotein of flavivirus, and the non-structural NS3 and NS5 encode essential enzyme activities for viral reproduction. The E protein is divided into three discernible domains . Domain I is involved in the envelope structure organization, and Domain II and Domain III are related to the monomers interaction and receptor binding, respectively  are of utmost importance for virus clearance. Cytotoxic CD8+ T cells are critical to eliminate virus-infected cells while CD4+ T cells provide help to cytotoxic CD8+ T cells and antibody production , 6. DNV-22.1in vitro (®), a phenomenon possibly related to ADE , the persistence of biomarkers of ZKV infection  suggest that some cells may be chronically infected. The wide distribution of types and anatomical locations of cells permissive for ZKV infection, sometimes beyond the easy reach of antibodies , suggest that a cell mediated immune response will be critical for immune surveillance of chronically infected cells. While there can be little doubt that a ZKV vaccine stimulating a neutralizing antibody response will be a key resource in limiting viremia during the acute phase of ZKV infection, there are some concerns regarding the exact nature of the antibody response provoked. The exact pathological mechanism which drives Guillain–Barré syndrome (GBS) remains unknown although there seems to a general consensus that antiglycolipid antibodies play an important role, although not every GBS patient develop this type of antibody. As discussed earlier, there is an increased incidence of GBS associated with ZKV infection , 19, butin vitro . This isd to ADE . This isd to ADE . Their rd to ADE , 28–31. d to ADE . Preclind to ADE , 34.Taken together, these findings suggest caution in needed in the development of whole protein ZKV vaccines where evoked antibody responses that are not neutralizing may possibly enhance infection or be pathogenic  or could facilitate infection of maternal–fetal interface tissue.2.2Given the concerns with antibody-inducing flavivirus vaccines, one possible alternative would be to harness the power of the T cell immune response in protecting against flavivirus infection, as mentioned above. A recent report has shown that CD8+ T cell prevent antigen induced antibody-dependent enhancement of Dengue disease in a murine model and several studies have identified DNV T cell epitopes appropriate for inclusion in a T cell-based vaccine , 35–38. 2.3In chronic HIV infection there exists a reservoir of latent, transcriptionally silent viral infection within the resting memory CD4+ T cell compartment and specific myeloid lineage cells  . ThA recent murine model study has demonstrated the potential importance of the CTL response to ZKV infection where H-2D restricted CTL epitopes were identified . StudiesFlaviviruses mutate in response to immune system pressure, both by antibodies and T cells. It has been reported that HLA class I-binding residues of a CD8+ T cell epitope encompassing the conserved catalytic site of DNV NS3 protease suffer variation that can abrogate HLA class I binding, suggesting evasion of DNV from a specific CD8+ T cell response by antigenic drift . Antigen2.4Human class I epitopes have not yet been formally identified for ZKV. Some authors have published ZKV MHC class II epitope prediction based on MHC binding search engines alone , 56, 57.We generated the predicted ZKV epitope list using the sequence of ZKV Strain H/PF/2013 (GenBank Accession number: KJ776791.2) . This stIn order to maximize matching known DNV class I epitopes against the ZKV sequences, we were indiscriminate with respect to the DNV strain sequences. We used epitope sequence data from all of DNV strains 1–4, as downloaded from IEDB. Alignments between predicted ZKV epitopes and DNV were calculated using MAFFT  and webPA recent study by Stettler  indicate2.4.1X-ray crystallography can be used to generate a PDB file containing a complete mathematical representation of the three-dimensional properties of a protein . SoftwarSE) which codifies the extent to which a free energy change will occur after CTL escape at that epitope , and in silico prediction correlated with promiscuity in HLA-binding assays and frequency of T cell recognition by exposed individuals , BeH819015 (Genbank accession number KU365778.1), BeH815744 (Genbank accession number KU365780.1), BeH819966 (Genbank accession number KU365779.1), SPH2015 (Genbank accession number KU321639.1), and SSABR1 (Genbank accession number KU707826.1), isolated in Brazil; and the H/PF/2013 strain (Genbank accession number KJ776791.2) isolated in French Polynesia were assembled and aligned with Clustal W (MegAlign, DNASTAR, Madison, WI, USA, Figure 2.7H-2D-restricted class I epitopes, when injected intradermally without adjuvants, produce a weak immune response in C57BL/6 mice. Methods have been described for eliciting immune responses to class I. For example, the target epitopes are linked together as a “string of beads” . In anotThe use of a biodegradable, PLGA microsphere-based vaccine delivery platform allows one or more unmodified peptides to easily be incorporated into the vaccine formulation . The limIn contrast to previous studies which incorporated only a single peptide epitope in spheres , we showThis is an important consideration, especially because the HLA restricted nature of the class I epitopes being delivered will require the development of a “master vaccine” containing enough different peptide epitopes to cover a target population.The fact that the majority of the epitopes listed in the first four rows of Tables 3The search for rapid development of safe and effective vaccines against ZKV is a global public health emergency. Testing multiple vaccine platforms in parallel may speed up and increase the likelihood of finding a good vaccine. We have proposed a rationale for ZKV epitope selection and design of T cell epitope-based vaccine against ZKV virus. Selection of candidate ZKV structure-constrained HLA class I epitopes able to bind an array of HLA class I supertypic molecules, and promiscuous class II T cell epitopes capable of binding to multiple HLA class II molecules could provide wide HLA and population coverage for such a vaccine which could be delivered using the synthetic, adjuvanted microsphere vaccine as outlined above or other techniques for epitope immunization that we discussed.This study was carried out in accordance with the policies and procedures established by the WIRB Institutional Review Board with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the WIRB.SE calculations for class I epitopes.RR, EC-N, DR, and PH wrote the manuscript. All authors reviewed the manuscript. All authors directly participated in the research described with EC-N and DR performing modeling predictions of class II epitopes and AM, TO, SC, and RR performing class I epitope identification and RR is CEO of Flow Pharma, Inc. RR, AM, TO, SC, and CH have received compensation in the form of cash and/or stock from Flow Pharma, Inc. All other 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.