In the small intestine, epithelial cells are derived from stem cells in the crypts, migrate up the villus as they differentiate and are ultimately shed from the villus tips. This process of proliferation and shedding is tightly regulated to maintain the intestinal architecture and tissue homeostasis. Apoptosis regulates both the number of stem cells in the crypts as well as the sloughing of cells from the villus tips. Previously, we have shown that villin, an epithelial cell-specific actin-binding protein functions as an anti-apoptotic protein in the gastrointestinal epithelium. The expression of villin is highest in the apoptosis-resistant villus cells and lowest in the apoptosis-sensitive crypts. In this study we report that villin is cleaved in the intestinal mucosa to generate a pro-apoptotic fragment that is spatially restricted to the villus tips. This cleaved villin fragment severs actin in an unregulated fashion to initiate the extrusion and subsequent apoptosis of effete cells from the villus tips. Using villin knockout mice, we validate the physiological role of villin in apoptosis and cell extrusion from the gastrointestinal epithelium. Our study also highlights the potential role of villin’s pro-apoptotic function in the pathogenesis of inflammatory bowel disease, ischemia-reperfusion injury, enteroinvasive bacterial and parasitic infections. The small intestinal (SI) epithelium forms the largest and most significant barrier allowing the selective absorption of nutrients, electrolytes and water while maintaining a strict and effective barrier against intra-luminal toxins, antigens and enteric bacteria. SI epithelial cells are tightly adherent cells attached to each other and the extracellular matrix resulting in an architecture that fulfills the protective barrier function of the gut. This tissue composition in turn is maintained by the stringent regulation of cell number within the epithelium by a process that balances cell proliferation with cell death. In all mammalian small intestine, new epithelial cells are generated by the stem cells of the crypts of Lieberkühn every 2–6 daysSalmonella typhimurium, Cryptosporidium parvum and Bacteroides fragilis; exposure to bacterial lipopolysaccharide (LPS), tumor necrosis factor (TNF), indomethacin and Toll-like receptor 3 agonists; ischemia and ischemia-reperfusion injury, burn injury, trauma, increased lymphatic pressure; and in cocaine- or atropine-induced villus contraction7891011121314151617197Pathological epithelial cell shedding is associated with several disease states including inflammatory bowel disease (IBD), bacterial infections such as 2-terminal fragment of villin is pro-apoptotic and is expressed exclusively in the epithelial cells near the villus tips. Together with our previous study, we demonstrate here that while the anti-apoptotic function of full-length villin is physiologically important for maintaining the resistance to apoptosis in the post-mitotic villus cells, the cleaved villin’s pro-apoptotic function is required for intestinal cell turnover and regulates the sloughing of cells from the villus tips. In this study, we provide a molecular and physiological basis for villin as both an inhibitor but also an effector of apoptosis. Additionally, we describe a molecular mechanism for cell extrusion that is regulated by an epithelial cell specific actin regulatory protein, villin. Since epithelial cell shedding in humans is morphologically similar to that observed in mice, our study also provides novel insight into the physiological function of villin in the human GI tractIt has been shown that one of the first events in cells destined to undergo extrusion is redistribution of actin and actin associated proteins2224All methods were performed in accordance with the relevant guidelines and regulations. MDCK Tet-Off cells expressing super enhanced yellow fluorescent protein (SEYFP)-tagged wild-type (VIL/WT) or mutant villin (VIL/S4-S6) were transfected by using Lipofectamine 2000. Transfected cells were cultured in Dulbecco’s modified Eagle’s medium containing 100 μg/ml G418 sulfate, 100 μg/ml hygromycin B, and 10% fetal bovine serum. Cells expressing cerulean tagged VIL/S4-S6 were cultured in Dulbecco’s modified Eagle’s medium containing 100 μg/ml zeocin and 10% fetal bovine serum. To repress the expression of villin gene in MDCK Tet-Off transfected cells, cells were cultured in the presence of 10 ng/ml doxycycline as described previously2 and COOH-termini of villin (V-20 and C-19 respectively) were purchased from Santa Cruz biotechnology and Immunotech; the actin and tubulin antibodies were purchased from BD Transduction laboratories. Antibodies against cleaved caspase-3 were purchased from Cell Signaling.Monoclonal antibodies against NHSmall intestinal crypts were isolated and cultured in Matrigel in the presence of conditioned media containing EGF, R-spondin, N2 supplement, B27 supplement and Noggin to generate epithelial enteroidsE. coli BL21 cells and purified as described beforeFull-length and truncated villin proteins were cloned in pGEX-2T and were expressed in Cerulean tag and COOH-terminal fragment of villin (S4-S6) were inserted sequentially into the pBudCE4.1 vector. The cerulean tag was amplified, restricted and inserted into the NotI and KpnI site, as described previously2+ (0–200 μM) and F-actin.The severing activity of full length (VIL/WT) and truncation mutant (VIL/NT) of villin was determined by analyzing the rate of decrease in fluorescence of pyrene labeled actin as described beforeAll experimental protocols were approved by IACUC . Villin knockout mice were generated as described previouslyCells were subjected to apoptotic stress by incubating with camptothecin (CPT)  as described previouslyStatistical analysis was performed using the two-tailed Student’s t test; p was based on upaired samples and unequal variance. The error bars are the measured standard error (S.E.) of mean.In mouse intestinal epithelium villin functions as an anti-apoptotic protein to regulate GI homeostasis24342-terminal fragment (VIL/NT) or the COOH-terminal fragments (VIL/CT) of villin  hijack cell extrusion to invade the gut epitheliumin vivo such as in restitution and repair of the epithelium. Increased villin proteolysis following intestinal infection with the parasite Giardia duodenalis results in the accumulation of the COOH-terminal fragment instead of the NH2-terminal fragmentGiardia infection. We suggest that the anti-apoptotic function of the COOH-terminal fragment may play a role in wound repair and re-epithelialization of the gut following such enteric infections.In the intestine, defective apical extrusion signaling has been shown to contribute to more aggressive tumor hallmarks2-terminal pro- and a COOH- terminal anti-apoptotic fragmentC. elegans suggesting that such mechanisms may not be an exception but the prevailing mechanism in multicellular organismsin vivo villin is cleaved by meprin to generate the actin-severing NH2-terminal fragment to induce cell extrusion from the villus tips.The closest homolog of villin, gelsolin is cleaved by caspase-3 to generate an NHIn summary, our study demonstrates that the expression of full-length and cleaved villin correlates with the cell’s position along the crypt-villus axis and determines the ability of the cells to undergo apoptosis and extrusion. Our study also describes for the first time a physiological molecular mechanism to regulate extrusion from the villus tips. This process appears to be uniquely adapted to the epithelial cells, using an epithelial cell specific actin regulatory protein namely, villin. Further studies will provide greater insight into the complex regulation of cell survival, cell death and cell extrusion that is required for the maintenance of intestinal epithelial architecture and homeostasis.How to cite this article: Wang, Y. et al. Both the anti- and pro-apoptotic functions of villin regulate cell turnover and intestinal homeostasis. Sci. Rep.6, 35491; doi: 10.1038/srep35491 (2016).


Objective: Interaction between genetic and environmental factors is considered as major factors in Schizophrenia (SCZ). It has been shown that dopaminergic and noradrenergic neurotransmission dysfunction play an essential role in the SCZ pathogenesis. This study aimed to find the impact of functional 19-bp insertion/deletion (ins/del) polymorphism in dopamine beta-hydroxylase (DBH) gene on SCZ risk in a sample of Iranian population.Method: This case-control study was conducted on 109 SCZ patients and 116 matched healthy subjects. Genomic DNA samples were extracted from peripheral blood cells using salting out method. Genotyping of 19-bp ins/del DBH polymorphism was done using Polymerase Chain Reaction (PCR) method.Results: Neither the overall chi-square comparison of cases and controls , nor the logistic regression analysis  showed any association between DBH 19-bp I/D and SCZ in a sample of southeast Iranian population.Conclusion: Overall, our results did not support an association between DBH 19-bp I/D polymorphism and risk/protection of SCZ. Schizophrenia (SCZ) is a serious mental disorder characterized by a breakdown of thinking and emotions, with a loss of contact with reality . It affeDopamine beta-hydroxylase (DBH) is an intracellular enzyme that catalyzes the conversion of dopamine to norepinephrine . DBH knoA 19-bpins/del polymorphism, located 4.5 kb upstream of the transcriptional start site of DBH gene, has been shown to have a strong association with DBH enzymatic activity , 17. A sSome studies have evaluated the impact of 19-bp ins/del polymorphism of DBH gene in SCZ -23. To tThis case-control study was done from January 2014 to June 2015 on 109 unrelated schizophrenic patients who referred to Baharan Psychiatric Hospital . One hundred sixteen age and sex matched normal subjects free from any signs of neuropsychiatric disorders and unrelated to each other and the patients were selected as controls. SCZ diagnosis was based on DSM-IV-TR  Criteria. The local Ethics Committee of the Zahedan University of Medical Sciences approved the project, and written informed consent was obtained from all participants. Two milliliter of venous blood was drawn from each participant into EDTA, containing tube and genomic DNA, and extracted using salting out protocol; the samples were stored at -20 ˚C until use . GenotypStatistical AnalysisStatistical analysis was done by statistical package SPSS 18 software. Data were analyzed using independent sample t-test or χ2 test according to the data. Odds ratio (OR) and 95% confidence intervals (95% CI) were calculated from logistic regression analyses to determine the possible association between the variants and ALL. A p-value of less than 0.05 was considered as statistically significant.The study group consisted of 109 SCZ patients  and 116 healthy participants . No significant difference was detected between the groups regarding age (p = 0.134) and sex (p = 0.88(.The genotype frequency of the DBH19-bp ins/del variant was tested for Hardy-Weinberg equilibrium (HWE) in the cases and controls separately. The genotypes in cases  and controls  were in HWE.Dysfunction of the dopaminergic and noradrenergic systems has already been well documented to be associated with SCZ . The genIn agreement with our findings, Hui et al.  found noIt has been shown that the del allelic and genotypic frequencies of DBH were significantly lower in controls than in patients with the first-episode of schizophrenia (FES), but controls were not significantly different from chronic schizophrenics (DBH plays an important role in the noradrenergic system. This enzyme is located within vesicles of central noradrenergic and adrenergic neurons and neurosecretory cells where it catalyzes the conversion of DA to NE , 31. TheRecently, Long et al.  have fouThe discrepancy in findings between studies may be due to genetic and environmental variances among the different populations being investigated.There are some limitations in the current study. One of the limitations of this study is its relatively small sample size, so we could not perform subgroup analyses. Another limitation of our study is that we investigated only one variant of DBH gene. Thus replication with larger sample size and other variants of DBH is highly recommended.In conclusion, the findings of this study showed that a 19-bp ins/del functional polymorphism of DBH gene did not affect the risk of schizophrenia in a sample of Southeast Iranian population. Conducting similar studies with larger sample sizes and different ethnic populations are needed to confirm the findings.


Allium macrostemon Bunge, wild onion) with leaves showing chlorotic stripes was collected in Saitama Prefecture, Japan. Genome sequencing showed that it was infected with shallot latent carlavirus. The genomic sequence of this virus is reported for the first time from wild onion.A wild Japanese garlic plant ( Allium macrostemon Bunge) is the most widely distributed wild onion plant in Japan. Only scallion mosaic potyvirus has previously been reported from Japanese garlic  found in this plant and compare it to those of SLV isolates reported in earlier studies   were used to identify the virus. A part of the SLV genome was identified by RT-PCR when SLV-specific primer pairs with a NotI restriction site were used. These primer pairs amplify the region from the middle of the TGB1 coding region to the 3′ end of SLV genome. The RT-PCR products were cloned into the NotI site of the plasmid pZErO-2 vector. The nucleotide sequences of the region in three plasmid clones were determined, and this confirmed that the sequences were nearly identical to those reported for SLV. Therefore, we focused on this virus in the subsequent experiments.Sap from specimen WOST87 was manually inoculated to leaves of The cDNAs of the remainder of the genome  were ampliﬁed and cloned similarly. The nucleotide sequences of at least three plasmid clones were determined for each region. The clone sequences with more than 400-bp overlapping regions were assembled to obtain the whole-genome sequence. The RT-PCR fragments were also directly sequenced. The whole-genome sequence, excluding a 23-nt 5′-end primer sequence used for amplifying the genome, was 8,338 nt in length.Carlavirus should have less than about 72% shared nucleotide identity (or 80% amino acid identity) in their RdRp and CP coding regions. Hence, our virus is an isolate of SLV, although, as its sequence identities were so different from those of previously reported SLV isolates, we consider our virus to be a distinct strain. This is the ﬁrst report of SLV collected from wild onion in Japan.The nucleotide and amino acid identities for the RNA-dependent RNA polymerase (RdRp) and coat protein (CP) coding regions of WOST87 and SLV , were calculated using EMBOSS Needle (LC279526.The sequence has been deposited in GenBank/EMBL/DDBJ under the accession number


Post-stimulatory cortisol was defined as primary endpoint and secondary endpoints include basal cortisol levels, post-stimulatory cortisol increments and the fold increase of cortisol one hour after stimulation. Sickle cell patients had an impairment of adrenal function despite no significant difference between patients’ and controls’ for basal or post-stimulatory cortisol levels. In fact, one patient in two failed to achieve a two-fold increase in cortisol levels after stimulation (5/10) as opposed to 1 in 10 in the control population (1/10), P = 0.070. The percent increment of cortisol after stimulation was lower in patients versus controls . Relative adrenal insufficiency is frequent in sub-Saharan adult patients with sickle cell disease despite normal basal cortisol levels. Our results suggest that adrenal function require further investigation during SCD crises as these represent an important stress and may worsen the prognosis.Rheological modifications observed in sickle cell anemia are associated with ischemic complications that can cause target organ functional impairment. The objective was to investigate adrenal function of adult patients with sickle cell disease. In this cross-sectional study conducted in a tertiary referral hospital of the capital city of Cameroon, we enrolled ten crisis-free adult patients with sickle cell disease (SCD) and ten age- and sex-matched healthy individuals. We assessed adrenal function by testing basal cortisol levels and 60 min after tetracosactide (Synacthen Sickle cell disease (SCD) is an autosomal recessive disease that results from a point mutation on the beta-globin molecule leading to erythrocyte rigidity and increased episodes of vaso-occlusive crisis . Under pCrisis-free adult (> 18 years old) patients with SCD were enrolled over a period of seven months (June-December 2007). The study was conducted in Yaoundé Central Hospital, one of the four tertiary reference hospitals in the capital city of Cameroon. Corticosteroid administration in the last three months preceding the investigation, recent infection, coagulopathy, thrombocytopenia, ongoing treatment including medications that could impact on adrenal function  and recent episode of vaso-occlusive crisis were exclusion criteria. Age- and sex-matched healthy volunteers with normal hemoglobin genotype (HbAA) were recruited through advertisement among the general population as control individuals. Written informed consent was obtained from all participants. The study was approved by the institutional review board and the National Ethics Committee. Anthropometric and clinical data were collected via a standard questionnaire and clinical examination.® 0.25mg/ml, Sigma-tau, Rome, Italy) and 60 min after injection for assessment of plasma cortisol levels [® 2000 Immunoassay System, Siemens, Los Angeles, CA, USA). The detection sensitivity of the method is 5.5 nmol/l and intra- / inter-assay coefficient of variation obtained from two series of samples analyzed daily in duplicate during 20 days were < 5%Adrenal function was assessed after an overnight 12-hour fast. Blood sample was drawn from the antecubital vein prior to an IM injection of 0.25μg of tetracosactide  was calculated to detect a 20% difference from a mean plasma cortisol level of 430.40 nmol/l. The chosen cut-off value for type I was 5% to reach an 80% power using 95% confidence interval (CI). The primary endpoint in this study was plasma cortisol level 60 min after stimulation and secondary endpoints include basal cortisol level, post-stimulatory incremental cortisol variation and cortisol fold increase. The post-stimulatory incremental cortisol variation (?cortisol) was estimated as the proportion of the difference between cortisol levels 60 min after stimulation and basal cortisol values. Statistical tests were performed two-tailed using SPSS for windows version 17.0 . Results with P value < 0.05 were considered significant.Statistical differences between groups were determined by independent samples P = 0.080). Patients had on average 3 vaso-occlusive crises within the last twelve. SCD patients were significantly leaner than control individuals (P = 0.030). None of them had received a blood transfusion in the last 12 months. The characteristics of the study population are shown in P = 0.900). Patients’ mean post-stimulatory cortisol value was lower than control participants although the difference was not significant (P = 0.090). However, the incremental cortisol variation after tetracosactide stimulation was significantly lower in the presence of SCD . Overall, five out of ten (50%) patients failed to achieve an optimal adrenal stimulation by reaching a two fold increase of basal plasma cortisol levels.Twenty crisis-free SCD patients and healthy volunteers were included in the study with a 1:1 ratio and a 60% proportion of male in each group. Systolic and diastolic blood pressure were similar in patients and controls while patients’ body fat composition tended to be lower in the presence of SCD  as well as an excess of protein breakdown in African-American patients with SCD compared to control in individuals . InteresFurthermore, it is apparent that patients failed more often to double their cortisol levels after stimulation and the median increase in cortisol values was lower than in individual carrying normal hemoglobin genotype. Basal plasma cortisol level has previously been reported to be lower in SCD children compared to both individuals with HbAA and HbAS genotypes . In factThe limitations of our study include the small sample size; although appropriate and well powered, a bigger study population may have enhanced the magnitude of estimates. Also, we do not report on basal plasma ACTH levels that could reveal the primary or secondary origin of the adrenal function impairment; 3. The proportion of SS hemoglobin in patients has not been assessed; this may have given some clues on the association with the frequency of vaso-occlusion events thus aid in understanding the relationship with the resulting adrenal insufficiency . FinallyOur findings suggest that relative adrenal insufficiency is not a rare event in crisis-free adult patients with SCD. We believe that this study may pave the way to further investigations of adrenal function during vaso-occlusive crisis and to subsequent studies aiming at unraveling the interplay between iron-overload, acute low-grade inflammation, oxidative stress and adrenal insufficiency in SCD patients in the presence or not of vaso-occlusive events.The propensity to thromboembolic events and iron deposition could potentially affect adrenal function in patients with SCD;There is potentially an exacerbation of adrenal dysfunction in patients with SCD with subcritical hypoadrenalism during sickle cell crisis which could unravel a clinically overt adrenal insufficiency;There is a paucity of data comparing cortisol response after stimulation in crisis-free patients with SCD and healthy (non-SCD) individuals.This study sought to compare stimulated cortisol response of crisis-free patients with SCD to age- and sex-matched non-SCD controls;We find a relative difference in adrenal response in crisis-free patients with SCD compared to the control population but not to a level suggesting a higher tendency to adrenal insufficiency in patients with SCD;These data suggest that further investigation in a larger population and comparisons of these findings with those in SCD patients during vaso-occlusive crisis is needed for a better understanding of the potential disease impact on pituitary-adrenal function.The authors declare no competing interests.All authors have read and agreed to the final manuscript.


Brain aging is a complex and heterogeneous process characterized by the selective loss and preservation of brain functions. This study examines the normal aging effects on the cerebral cortex by characterizing changes in functional connectivity using resting-state fMRI data. Previous resting-state fMRI studies on normal aging have examined specific networks of the brain, whereas few studies have examined cortical-cortical connectivities across the entire brain. To characterize the effects of normal aging on the cerebral cortex, we proposed the Pearson functional product-moment correlation coefficient for measuring functional connectivity, which has advantages over the traditional correlation coefficient. The distinct patterns of changes in functional connectivity within and among the four cerebral lobes clarified the effects of normal aging on cortical function. Besides, the advantages of the proposed approach over other methods considered were demonstrated through simulation comparisons. The results showed heterogeneous changes in functional connectivity in normal aging. Specifically, the elderly group exhibited enhanced inter-lobe connectivity between the frontal lobe and the other lobes. Inter-lobe connectivity decreased between the temporal and parietal lobes. The results support the frontal aging hypothesis proposed in behavioral and structural MRI studies. In conclusion, functional correlation analysis enables differentiation of changes in functional connectivities and characterizes the heterogeneous aging effects in different cortical regions. The blood-oxygenation-level-dependent (BOLD) signal measured through fMRI reflects hemodynamic changes resulting from local neural activity , 2. The During the resting state, subjects are asked not to perform any explicit cognitive tasks. All spatial patterns of neural activity observed through resting-state fMRI reflect internal activities of the brain . Fluctuafi and ri are the time-courses (TCs) in given pixels, and μf and μr are the average values of the TC observations fi’s and ri’s  with m = 51. We generate 200 simulation replicates, with each simulation dataset of size n = 100. We set μ(t) = )T and v(t)ϵkij following a normal distribution with mean zero and variance Ckl; k,l = 1,2} by Bessel correlation function of the first kind and Matérn correlation function with parameter  in Scenario 1 and  in Scenario 2 [ξri} are generated from N , where λr is the eigenvalue of C. Then, we obtain the observations tj) = (X1(tj),X2(tj))T, ϵij = T.We perform simulation comparisons to examine the finite sample performance of the proposed estimate enario 2 . The mFPThe simulation results, summarized in Besides applying TCC to the resting-state fMRI data, we obtain the CCC matrices of all ROI pairs for the young and the elderly groups as shown in To our knowledge, this is the first report on the effects of aging on cortical-cortical functional connectivity. The effects were studied using novel PFCorr statistics-based functional connectivity definitions. Our results showed that the connectivities between the frontal and posterior lobes increased in the elderly group, particularly the connectivity between the frontal and temporal lobe. By contrast, the inter-lobe connectivities among the posterior lobes decreased in the elderly group. The connectivity between the temporal and parietal lobes displayed the strongest negative aging effect. These findings suggest that the aging effect on cortical-cortical functional connectivity is heterogeneous across the lobes.The essential question regarding the effects of aging on the brain is whether the aging patterns are heterogeneous across the lobes. From MRI data, scholars have reached a consensus on the aging effect that the effect of aging on the cerebral cortex and white matter is heterogeneous –41. ThesResearchers have proposed brain plasticity and compensation to explain the changes in normal aging , 42. AccThe present study demonstrated that the aging effects on inter-lobe functional connectivity can be classified into two main effects: namely the positive (increased) aging effect and the negative (decreased) aging effect. The aging patterns of the frontal and posterior lobes substantially differ. Inter-lobe functional connectivity among the posterior lobes showed a predominantly negative aging effect. By contrast, inter-lobe functional connectivity between the frontal and posterior lobes presented a predominantly positive aging effect, possibly because of the enhanced connectivity of the frontal lobe necessary for maintaining normal brain function in elderly adults. Our finding demonstrated that in the absence of a specific cognitive function, the frontal lobe exhibited strong dynamic cortical-cortical connections. In other words, the age-related changes in resting-state connectivity support the frontal-aging hypothesis.This study has some limitations. First, this was a cross-sectional comparison study between two age groups. A longitudinal study observing life-long aging effects on the same subjects will be more accurate; however, performing such a study is difficult. Second, this study focused on the functional connectivity of only the cerebral cortex. The aging effects on the functional connectivity of the limbic system and subcortical regions were not studied. Third, given the limited sample size  we can only divide the subjects into two groups, i.e. 40 subjects in the young group and 30 subjects in the old group, to achieve statistical power. Future studies could explore a division into a greater number of age groups by acquiring much larger datasets across the lifespan; this could give us greater resolution to identify differences between specific ages. Fourth, our data includes elderly brains which were atrophy. The normalization of the atrophy brain to the template has been a technical issue in the neuroimaging filed. The normalization process mainly relies on the segmentation and spatial normalization via different algorithms. SPM, DARTEL, and FSL tools are the most frequently used. The previous comparative study indicated that the unified segmentation/normalization of SPM8 revealed the largest age-related differences and may overestimate the aging effect on the brain volume . This wiIn conclusion, we propose a novel functional correlation approach for measuring cortical-cortical functional connectivity. Using this approach, we characterized the heterogeneous aging effects on functional connectivities of the cerebral cortex. Given the characteristic patterns in a normal aging brain, the proposed approach is potentially useful for investigating cortical-cortical functional connectivities in age-associated brain diseases, such as Alzheimer's disease and Parkinson's disease.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(RAR)Click here for additional data file.S4 File(ZIP)Click here for additional data file.


Global DNA methylation of long interspersed nucleotide elements (LINE-1) in leukocytes has been suggested to be a risk factor for a few cancers. There has been no report of LINE-1 methylation in leukocytes as a risk factor for aggressive prostate cancer at diagnosis and prognosis after treatments. In this study, we measured the leukocyte DNA methylation of LINE-1 in 795 PCa patients and compared the methylation levels across different clinical subgroups. We then determined the association of LINE-1 methylation in leukocytes with clinicopathological variables at diagnosis using logistic regression analysis and biochemical recurrence in patients receiving active treatments (prostatectomy and radiotherapy) using Cox proportional hazard model after adjusting for age, BMI, smoking status, pack year, D’Amico risk groups, and treatments. Overall, the DNA methylation of LINE-1 was not associated with the risk of being diagnosed with high-risk prostate cancer or the risk of biochemical recurrence upon active treatments. Future studies are warranted to investigate other types of repetitive element methylation and longitudinal changes of global methylation in relation to prostate cancer risk and prognosis. Prostate cancer (PCa) represents the most common cancer and the third leading cause of cancer death in American men . AlthougEpigenetic changes including DNA methylation, histone modification and microRNA regulation play important roles in PCa development and progression . DNA metGlobal DNA methylation predominantly occurs within highly repetitive DNA sequences, such as long interspersed nucleotide elements (LINE-1) and short repetitive sequences Alu repeats , 16. TheThe characteristics of the 795 PCa patients and the LINE-1 methylation level stratified by these characteristics are shown in Table We then analyzed the association of the methylation of these CpG sites with BCR in patients receiving active treatment (prostatectomy and radiotherapy) Table . We dichDNA methylation plays important role in tumorigenesis and progression. Methylation profile is an inheritable feature that could affect genomic instability and gene expression. To our knowledge, this is the first study to evaluate the association of LINE-1 DNA methylation in peripheral blood leukocytes with the aggressiveness of prostate cancer. We did not find significant associations between LINE-1 methylation and presenting with high risk PCa at diagnosis or PCa prognosis.LINE-1 is a highly abundant repetitive sequence in human genome with about half a million copies and accounting for nearly 18% of human genome . The thrOne interesting observation was that current smokers may have lower LINE-1 methylation than never smokers , and progression . The MD Anderson Tumor Registry conducts annual vital status follow-ups for all cancer patients. Biochemical recurrence was defined as a serum PSA level of at least 0.2 ng/ml with a second confirmatory PSA level of at least 0.2 ng/ml for patients who undergo a radical prostatectomy  or with CGTGGTGCGTCG, which contained three CpG sites. Bisulfite-treatment of DNA converts all unmethylated cytosine to uracil, but leaves methylated cytosine intact. The ratio of the signal from cytosine (that signifies a methylated cytosine) to the sum of cytosine plus thymidine for each specific CpG site was termed β value as a parameter of methylation percentage at that site. The overall methylation was presented as the average methylation measured for all three sites. The PCR primer and pyrosequencing probe sequences were: TTTTGAGTTAGGTGTGGGATATA (forward), Bio-AAAATCAAAAAATTCCCTTTC (reverse), and AGTTAGGTGTGGGATATAGT [PCR and pyrosequencing of LINE-1 were performed as previously described . In brieGATATAGT . PCR reaSimilar number of GS 6, 7 and 8 cases was placed in each run and different batches were treated as a nuisance covariant in our analysis to control batch effect. We compared the methylation of three individual CpG sites and the average of the three CpG sites of LINE-1 among patients with different clinical characteristics at baseline using analysis of variance (ANOVA). Multivariate logistic regression was applied to evaluate the odds ratio (ORs) and 95% confidence intervals (CIs) for analyzing the association of LINE-1 methylation with aggressiveness of PCa at diagnosis after adjustment of co-variates such as age, smoking and BMI. We then analyzed the association of CpG site methylation with the risk of biochemical recurrence (BCR) in patients receiving radical prostatectomy or radiotherapy using multivariate Cox proportional hazards model adjusting for age, BMI, smoking status, pack year, D’Amico risk groups, and treatments. Time to BCR was calculated from the date of definitive treatment  to the date of BCR or date of censor. The median follow-up time was 44.3 months.


Reconstructed annual streamflows using tree-ring chronologies provide useful information on moisture availability during the pre-historic period, but they have limitations in estimating high flows due to the upper bound on soil water holding capacity and trees’ metabolic growth limits. We propose a hybrid approach that uses tree-ring chronologies and climatic indices for improving high flows in 301 basins whose annual streamflows are modulated by ENSO and/or PDO. The hybrid decomposition approach relies on separating the moisture supply into the basin as outside-the-region moisture and within-the-region moisture with the former being estimated by SST indices and the latter being estimated by tree-ring chronologies. Analyses over the 301 stations from coterminous US show that the proposed approach improves the high flows and improves the overall error in the reconstructed streamflows. Potential utility of the improved reconstructed annual streamflows with improved high flows is also discussed. The high dependency between moisture availability and tree-ring chronologies have provided information on wet and dry periods by reconstructing streamflows over pre-instrumental periods8. While tree-ring chronologies have good skill in low flow estimation11, they have limitations in estimating high flows. Due to the upper bound on water holding capacity of soil, trees’ metabolic growth limits and increased cloud cover during wet years, high flows are not properly estimated using tree-ring chronologies14. Studies have shown that incorporating sea surface temperature (SSTs) along with tree-ring chronologies resulted in improved reconstruction of streamflow and wildfire synchrony17. Recent progress in understanding ocean-atmosphere interactions and the ensued teleconnection shows that there are well organized modes of interannual and interdecadal variability in climate that modulate these dominant moisture delivery pathways and has significant projections on continental scale rainfall and streamflow patterns19. Interannual modes such as the El Nino-Southern Oscillation (ENSO) resulting from sea surface conditions in the tropical Pacific Ocean primarily determine the interannual variability in precipitation over North America21. There are also other dominant decadal and interdecadal climatic modes such as Pacific Decadal Oscillation (PDO) and North Atlantic Oscillation (NAO) that putatively govern the interannual variability in climate over the North America22. To reduce the limitation of tree rings in reconstructing high flows22, proposed a hybrid reconstruction methodology using SST and tree-ring chronologies building upon the teleconnection signal with exogenous climatic conditions22. suggested separating the moisture contributed from exogenous climatic conditions  as “outside-the-region moisture supply” and moisture contributed from the same hydroclimatic region as “within-the-region moisture supply”. Since the study22 focused on the Southeast US (SEUS), outside-the-region moisture transport was estimated by El-Nino Southern Oscillation (ENSO) Index, Nino3.4, the within-the-region moisture contribution was estimated by tree-ring chronologies. Given the influence of outside-the-region moisture supply on streamflow is separately estimated by exogenous SST conditions, it partially improves high flow estimation that is contributed by SST sources. The hybrid reconstruction approach, relying on utilizing multi-proxies  for streamflow reconstruction, by22 improved overall skill and resulted in better above-normal flow estimates in comparison to the traditional tree-ring reconstructions in eight stations across the SEUS, which is a region with limited tree-ring chronologies. We perform here a national-scale hybrid streamflow reconstruction for 301 stream gauges  for the same. Given that SSA approach requires low-frequency mode identification for separating SST signals, such an approach will be difficult in the current study which considers PDO that has a periodicity of 16–20 years. The current approach is such simpler by explaining the role of SST predictors in influencing the Box-Cox transformed streamflow.Annual streamflow records provide crucial information on the available water for various uses, including water supply and irrigation and for designing reservoir size. Given little observed streamflow data is available before the 1930s in the United States, studies focused on gaining insights on annual moisture supply prior to the observational period using paleo-information24. Details on the variability explained by ENSO and PDO on the annual flows of the selected Hydro-Climatic Data Network (HCDN) basins is given in Supplemental Information (SI)  conditions are used as SST conditions that have been shown to significantly affect moisture transport over the CONUS Figs SI- and SI-22, 25. Figure 24. Both models showed insignificant skill in predicting observed flows  . For instance, existing naturalized inflow time series for basins with significant anthropogenic influences could be used with the proposed hybrid methodology for developing reconstructed flows, which could be used for capacity expansion of reservoir and for revising the current design yield considering the paleo-information. In fact, studies have shown the information from streamflow reconstruction improves reservoir sizing37 and provide better insight into future streamflow conditions in comparison to near-term climate change projections38. Potential to re-assess operational rule curves to incorporate environmental flows also exists using the augmented record40.Though the reconstruction was performed in virgin basins with relatively long data records from the CONUS, the hybrid reconstruction methodology is expected to provide significant benefits for basins with limited streamflow records as both SST anomalies and tree-ring chronologies are available for a longer period. Further, both these data sources extend beyond the available meteorological records , thereby ably supporting Prediction in Ungauged Basins (PUB) efforts41 is primarily estimated from ships and limited buoys. Thus, additional investigation is needed on how the uncertainty in SSTs propagates into streamflow reconstruction. One approach is to use available meteorological records from 1900s with a hydrologic model and compare with the reconstructed flows. Another approach is to replace the SST anomalies with the General Circulation Model (GCM) simulated SST time series from Coupled Model Intercomparison Project Phase 5 (CMIP5)42 with the hybrid methodology and compare the performance with the AM skill. However, both these additional validation efforts suffer from errors from the hydrologic model and from GCMs.Although the proposed hybrid methodology shows improvements in reconstruction skill, the methodology has limitations. Since the model estimates the outside-the-region moisture transport using SST anomalies, the proposed reconstruction could be applied only for basins that exhibit significant influence due to low frequency climatic signals. In addition, while the SST data is of good quality for a significant period (1940 – present), SST data prior to 194043. Similar nested reconstruction techniques could be considered to combine the reconstructed streamflows from the hybrid approach  and the traditional approach , thereby developing a long time series of reconstructed streamflows with different uncertainty over the entire period. Similarly, the proposed methodology also did not consider inter-site correlation as the effort here focused at the national scale. One could consider techniques44 that explicitly considers inter-site correlation with the proposed technique to accommodate for basins exhibiting significant inter-site correlation.Since the hybrid methodology considers SST data for reconstruction, it also depends on the length of the SST data, which is available from 1856 onwards. Thus, it potentially limits the length of reconstructed data. Dendrochronological studies have developed reconstructed hydroclimatic data using multi-species data having different uncertainty over different time intervals26 have shown that both these indices influence the hydroclimatology of the basins predominantly at the regional level. Further, we did not consider it for reconstruction due to non-availability of these indices dating back to 1856. Extending these indices back to 1856 and incorporating them in the proposed hybrid methodology of using multi-proxies could further improve the reconstructed streamflows for basins exhibiting influence with AMO and NAO.The inability of tree-ring chronology to capture the moisture availability during wet years due to soil and metabolic limits results in reduced interannual variability in the reconstructed annual streamflows. Given the limitation in quantifying wetter conditions, it is expected that the uncertainty in tree-ring chronologies to be larger in comparison to instrumental records in quantifying the moisture availability over the basin. So, our hybrid approach of using multi-proxies – SST and tree-rings combined – improves the streamflow reconstruction. We also considered other teleconnection indices such as North Atlantic Oscillation and Atlantic Multidecadal Oscillation in influencing the annual streamflows, but only fewer basins showed significant skill in influencing the annual streamflows. Studies46 and are considered as undeveloped/virgin basins with no impacts due to upstream storage or groundwater pumping. The tree-ring chronologies used for streamflow reconstruction in this study are from the National Atmospheric and Oceanic Administration (NOAA) International Tree Ring Data Bank (ITRDB)  Fig. . Tree riDB) Fig. ,2 are loDB) Fig. . Only HC24. ENSO conditions, denoted by Nino3.4, were obtained from 1856 from International Research Institute for Climate and Society (IRI) data library using the Kaplan’s SST41. Time series of PDO from 1856 was also obtained from the NCEP teleconnections database47 using Extended reconstructed SST (ERSSTv4)48. Both Kaplan’s and ERSSTv4 SST database utilize optimal interpolation methods to develop a unified global SST database with uncertainty bounds from 1856 to till date by combining SST observations from buoys and ships. For details see49. Basins were classified as ENSO and PDO affected if the Spearman’s rank correlation between the observed streamflow and Nino 3.4 and PDO indices respectively was significant at a 95% confidence level, and HCDN basins affected by neither were not considered for this study. Correlations between Nino 3.4 and PDO and annual streamflow are shown 52. SSA is a dimension reduction analysis which identifies periodic components of a time series by comparing lagged versions of the time series52. only considered the ENSO signal on streamflow which has a periodicity of three to seven years. In contrast, PDO has a periodicity of 10–20 years, making the signal difficult to identify, especially for HCDN basins with less than 30 years of data. Given the complications of PDO in this study, we pursued the approach presented in Fig. Figure  Fig. SI-. [see SI53. Using tree-ring principal components (PCs) explaining 90% of the variance and the transformed streamflow  utilizes only tree-ring chronologies identified as predictors for each site to explain all components of annual streamflow. Since tree ring chronologies across the sites exhibit high correlation, principal component analysis (PCA) was performed on the tree ring chronologies to obtain predictors for the regression modelAM (purple) Fig.  separateThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.Supplementary information file


There have been reports on stent-related vascular erosions about patients with benign or malignant stenosis of the esophagus who received endoscopic stent insertion for palliative intention for oral intake.A 61-year-old woman with esophageal cancer located in the middle part of esophagus was treated with esophagectomy. Two years following the surgery, malignant stenosis recurred in the esophagogastric anastomosis. A non-covered self-expanding metal stent (10 cm length with a diameter of 18 mm at expanded state) was inserted. Three months later, a massive hematemesis with subsequent hemorrhagic shock developed from the proximal end of the stent which resulted in the final diagnosis of arterioesophageal fistula on the left subclavian artery. An endovascular repair using a stent graft for the left subclavian artery via the right common iliac artery was performed and the patient remained well until discharge. Increase in the treatment of esophageal strictures by stent insertion increases the risk of stent-related vascular fistula. These complications should be considered in any patients with massive upper gastrointestinal bleeding. Standard therapy for early esophageal squamous cell carcinoma is surgical resection or neo-adjuvant chemoradiotherapy followed by resection. In advanced stages, palliative therapy such as endoscopic placement of a stent is a safe and effective procedure to relieve dysphagia . EsophagWe describe a complication of a self-expanding metal stent in a patient with recurrent malignant stenosis after esophagectomy due to esophageal carcinoma.A 61-year-old woman with an esophageal squamous-cell carcinoma at stage T2N0, in the middle part of the esophagus with no lymph node involvement was treated with an esophagectomy and gastric pull-up. Two years following the surgery, dysphagia developed and malignant stenosis recurred in the esophagogastric anastomosis. Because of patient disinclination, chemoradiotherapy was not done and a non-covered self-expanding metal stent (10 cm long with a diameter of 18 mm when expanded) was inserted. Three months later, severe hematemesis and hemodynamic shock developed. Endoscopic examination revealed massive hemorrhage from the proximal end of the stent. An endoscopic intervention to stop the bleeding was unsuccessful. Computed tomography (CT) and CT-angiography demonstrated an arterioesophageal fistula on the left subclavian artery in contact with the esophageal stent  and 2. TEsophagoarterial fistula is an infrequent complication after esophagectomy . The fir


A 16-year-old male presented to the emergency department (ED) with left-sided weakness. He described having a headache with dizziness for seven days prior to presenting with new onset weakness. The patient was from Central America with no known medical history and was unvaccinated. The examination was significant for flaccid paralysis with decreased sensation in the left upper and lower extremities, plus a left-sided facial droop. The cardiac examination revealed a 2/6 diastolic murmur, loudest at the left sternal border. A point-of-care ultrasound performed in the ED demonstrated a left atrial mass swinging into the left ventricle during diastole  and 2.Computed tomography angiography of the head led to a diagnosis of left atrial myxoma with embolism resulting in non-hemorrhagic occlusion of the right middle cerebral artery . After iCardiac tumors are a rare diagnosis, with a reported incidence of 0.01–0.02%.What do we already know about this clinical entity?Cardiac myxoma is a rare, benign tumor of the heart that predisposes patients to embolic phenomena involving cerebral or mesenteric arteries typically requiring surgical repair.What is the major impact of the images?Point-of-care ultrasound (POCUS) visualization of a left atrial myxoma, and subsequent computed tomography of a cerebral artery-filling defect, solidified our diagnosis of tissue embolus, which explained the patient’s constellation of symptoms and led to timely surgical consultationHow might this improve emergency medicine practice?This case highlights the utility of POCUS in the emergency department to quickly identify life-threatening ailments that may otherwise go undiagnosed.Documented patient informed consent and/or Institutional Review Board approval has been obtained and filed for publication of this case report.


Nanomaterials have gained tremendous significance as contrast agents for both anatomical and functional preclinical bio-imaging. Contrary to conventional medical practices, molecular imaging plays an important role in exploring the affected cells, thus providing precision medical solutions. It has been observed that incorporating nanoprobes improves the overall efficacy of the diagnosis and treatment processes. These nano-agents and tracers are therefore often incorporated into preclinical therapeutic and diagnostic applications. Multimodal imaging approaches are well equipped with nanoprobes to explore neurological disorders, as they can display more than one type of characteristic in molecular imaging. Multimodal imaging systems are explored by researchers as they can provide both anatomical and functional details of tumors and affected tissues. In this review, we present the state-of-the-art research concerning multimodal imaging systems and nanoprobes for neuroimaging applications. The success of medicine and healthcare is largely determined by the efficacy of treatment techniques. By incorporating functionalized nanoprobes and/or tracers with imaging systems, better results can be achieved for diagnosis and treatment of neurological diseases ,2. SpeciImaging systems serve as an excellent diagnostic tool by capturing the details of the disease and therapeutic process on injecting bio-chemical agents or nanoprobes. Several imaging techniques have been developed over time to study the effects of therapeutic agents . In manyThe most frequently used imaging systems in diagnostic applications include Positron Emission Tomography (PET), Computed Tomography (CT), Single-Photon Emission Computed Tomography (SPECT) and Magnetic Resonance Imaging (MRI). By definition, depending on the requirement, these modalities are appropriately chosen in specific combinations. Histed et al.  explainsAs previously mentioned, the role of theranostic agents is extremely important and cannot be ignored. In addition to fulfilling therapeutic requirements, they must also yield themselves to imaging and diagnostic requirements. Iron oxide nanoparticles ,12, goldOwing to the significant advances in nanoprobes, multimodal imaging and the plethora of works available in the field, there is an impending need to bring to order the latest trends, which is the focus of this paper. In this review, we classify the existing literature according to multimodal imaging system used. In doing so, we provide a comprehensive overview of the nanoprobes/tracers used for neuroimaging applications and the context in which they are used, classified as per the multimodal combinations as PET-CT, PET-MRI and SPECT-CT.This paper is subsequently organized as follows. In this section, we discuss the works employing PET-CT as the imaging modality for neurological applications with nanoprobes as contrast agent. The need for this integrated combination is directly related to the desire to overcome the shortcomings of using only one modality or multiple modalities independently. In this case, PET has an innate ability to determine the metabolic activity of tissues but cannot provide details at a high resolution. Although CT cannot provide for sharp differences in physiology, it is the perfect companion to the PET for high resolution anatomical details. This potent combination is therefore a good solution for imaging and lesion localization .18F–FLT) was used as the radiotracer for imaging using PET-CT as shown in 18F–FLT PET on the estimation of post-operative target volumes for radiotherapy was investigated. The authors concluded that post-surgery tumor volumes detected by 18F–PET are not always consistent with those obtained through MRI and that incorporation of 18F–FLT-PET significantly improves the delineation of target volume for radiotherapy.Zhao et al.  compares18F–Fluro-ethyl-tyrosine (18F–FET) to identify the total tumor volume in skull-base lesions for image-guided stereotactic radiotherapy with PET-CT. They also show the comparative advantages of the FET tracer for target identification in the therapy planning process. Unlike FDG-PET, better accuracy detection rates may be obtained by employing FET-PET. 18F–FET demonstrates a much lower sensitivity but a higher specificity compared to FDG. Furthermore, 18F–FET allows a precise estimation of cranial border tumors compared to FDG.Badakhshi et al.  presentsNonokuma et al.  presents11C–Methionine (MET-PET) was used as the tracer for experiment using PET-CT. This is because the uptake of Methionine corresponds to the presence of lesions in the brain following the coiling procedure. The authors highlight the importance of conducting such a diagnostic imaging following coiling failing as the lesions could go unnoticed.Deguchi et al.  presente18F–FFMZ-PET images. As can be seen, the tracer agent used in this work is fluoroflumazenil because it can be reliably used to predict the final infarct with relatively few false positives. Zhang et al. [Wright et al.  propose g et al.  highligh18F–FDG-PET-CT system. Hatzoglou et al. [18F–FDG-PET-CT, while also assessing the effectiveness of each in order to achieve the objective as shown ratio may improve the efficacy of FDG-PET-CT as the imaging modality for this application. Lapa et al. [68Ga–Pentixafor-PET-CT imaging thereby allowing for the quantification of Chemokine receptor-4 (CXCR4) and the identification of suitable patients for therapy. 68Ga–Pentixafor-PET-CT was chosen as the imaging agent because of its strong affinity for CXCR4, which is reportedly overexpressed in glioblastoma lesions.Knesaurek et al.  demonstru et al.  focused a et al.  investigAs the name suggests, this hybrid imaging technique combines the power and flexibility of PET and MRI to avoid the pitfalls caused by using each modality independently. Jadvar et al.  describeGuo et al.  examined2O as tracer for PET/ MRI on stroke patients to overcome the limitation of single modality (MRI) information in helping identify critically hypo-perfused tissue during thrombolysis trials. This augmentation of standard stroke MRI with PET data can pave the way for better patient stratification and personalized healthcare. As opposed to the sequential model, simultaneous data acquisition using 15O-H2O with PET/MRI was used to relate Cerebral Blood Flow (CBF) measurements of MRI data with that of PET data, although the basic stroke care itself remains unchanged. Korsholm et al. [Werner et al.  employedm et al.  assessesm et al.  used FDG11C diprenorphine (11C–DPN) was chosen as the radiotracer for their experiments to identify the regional endogenous opioid displacement from the opioid receptor. The objective of their work was to identify where in the pain-activated brain network opioid modulation occurs. Through their investigations, the authors establish the co-localized occurrence of responses in the thalamus and striatum-related pain processing. These signal changes in the thalamus were positively correlated indicating that the opioid neurotransmitter changes induced by pain make up a significant component of the fMRI signal changes.Anazodo et al.  discusse18F–FDG-PET-MRI data to analyze the metabolic and structural changes in patients suffering from torture-related post-traumatic stress disorder. FDG tracer-based PET images are used to study metabolism in the different brain regions in the patients. They observed that the post-torture trauma was inimical to the morphology and the functions of the brain which was well captured through the use of the PET-MRI hybrid imaging system.Zandieh et al.  employed52Mn was produced in their experiments. By virtue of overexpression of the protein, increased Mn incorporation can be used for selective signal enhancement using the MRI making them suitable for cellular imaging. Henriksen et al. [18F–FET-PET-DSC MRI for evaluating tumor metabolism, structure and blood volume in the study of the human brain. 18 F–FET is used as the nanoprobe for this application as it can be used to investigate the transport of amino acids, which is a valuable addition in the evaluation of patients with brain tumor.Lewis et al.  exploredn et al.  used 18FThe SPECT-CT hybrid combination is yet another popularly used imaging system in medical theranostics because of its superior performance in lesion characterization, representation and findings as reported in ,38,39. BRangger et al.  used ded123I-β-CIT-SPECT-CT to evaluate the condition of the rat midbrain dopaminergic pathway in the developed 6-hydroxydopamine model of Parkinson’s disease. In order to synthesize a model of Parkinson’s disease, 6-Hydroxydopamine (6-OHDA) was used to induce the degeneration of neurons in rats. The dopamine transporter radio ligand 123I-β-CIT was used to estimate the DAT density in the striatum of the lesion-induced rats. The termination of dopamine signaling is the sole responsibility of DAT. As the DAT transporter is present only in the plasma membrane of the dopamine neurons in the central nervous system, it is an excellent biomarker for this network.Bäck et al.  employed123I-β-CIT dopamine transporter binding in the mouse brain which can prove useful for ascertaining neuroprotective properties of drugs in mice. Unlike previous works, which have employed 123I-β-CIT for imaging operations in rats and monkeys, this work strives to do so in mice. 123I-β-CIT exhibits good correlation with the Binding Potential (BP) which is directly proportional to the density of DAT in equilibrium. It is BP which is used to assess the 123I-β-CIT ratios in the studies of mice. Further, 123I-β-CIT has several advantages to offer, the main reason being the higher binding ratios being reported in humans thereby increasing the signal to noise ratio while operating and reporting from the data on the mouse brain. Their work is the first to report the 3D kinetics of 123I-β-CIT in mice.Pitkonen et al.  investigIn addition to these studies, functionalized nanoprobes are also developed for neuroimaging applications for other multimodal imaging systems such as PET-Fluorescence and MRI-Fluorescence. Although the multimodal imaging systems and probes available today offer several benefits in the preclinical and clinical arenas, there are potential loopholes or scope for future research that may have to be taken into consideration. Literature ,46 suggeThis paper provided an overview of the latest developments in the field of multimodal neuroimaging using nanoprobes, in combination with functional and anatomical imaging. This provides an insight into various probes and tracers developed for preclinical imaging with multimodal approaches such as PET-CT (Positron Emission Tomography/Computed Tomography), PET-MRI (Magnetic Resonance Imaging), SPECT-CT (Single-Photon Emission Computed Tomography), etc. The applicability of these probes is well demonstrated through various aspects of functional imaging in brains through PET and SPECT, together with anatomical CT or MRI images. These applications include basic metabolism of brain, neurodegenerative diseases, lesions, tumors, aging, cognitive dysfunctions, drug effects, blood flow study and various others. Multimodal imaging in these applications allows for capturing different aspects of the brain process while also overcoming the shortcomings of unimodal systems, thus making them a popular choice. It can therefore be concluded that multimodal imaging systems with nanoprobes are well demonstrated in preclinical setup for neuroimaging applications and, together with the upcoming research models, the path for clinical diagnostic, therapeutic and theranostic applications is being paved.


Nature Communications 10.1038/s41467-017-02069-z, published online 08 December 2017Correction to: The original version of this Article omitted the following from the Acknowledgements:“This work was partly supported by the Research Council of Norway through its Centres of Excellence funding scheme, project number 262633, QuSpin.”This has now been corrected in both the PDF and HTML versions of the article.


This is however not a simple feedforward flow of information: there is a significant feedback from cortical cells back to both relay cells and interneurons in the dLGN. Despite four decades of experimental and theoretical studies, the functional role of this feedback is still debated. Here we use a firing-rate model, the extended difference-of-Gaussians (eDOG) model, to explore cortical feedback effects on visual responses of dLGN relay cells. For this model the responses are found by direct evaluation of two- or three-dimensional integrals allowing for fast and comprehensive studies of putative effects of different candidate organizations of the cortical feedback. Our analysis identifies a special mixed configuration of excitatory and inhibitory cortical feedback which seems to best account for available experimental data. This configuration consists of (i) a slow (long-delay) and spatially widespread inhibitory feedback, combined with (ii) a fast (short-delayed) and spatially narrow excitatory feedback, where (iii) the excitatory/inhibitory ON-ON connections are accompanied respectively by inhibitory/excitatory OFF-ON connections, i.e. following a phase-reversed arrangement. The recent development of optogenetic and pharmacogenetic methods has provided new tools for more precise manipulation and investigation of the thalamocortical circuit, in particular for mice. Such data will expectedly allow the eDOG model to be better constrained by data from specific animal model systems than has been possible until now for cat. We have therefore made the Python tool  pyLGN which allows for easy modification and evaluation of the a priori quite general eDOG model to new situations.On route from the retina to primary visual cortex, visually evoked signals have to pass through the dorsal lateral geniculate nucleus (dLGN). However, this is not an exclusive feedforward flow of information as feedback exists from neurons in the cortex back to both relay cells and interneurons in the dLGN. The functional role of this feedback remains mostly unresolved. Here, we use a firing-rate model, the extended difference-of-Gaussians (eDOG) model, to explore cortical feedback effects on visual responses of dLGN relay cells. Our analysis indicates that a particular mix of excitatory and inhibitory cortical feedback agrees best with available experimental observations. In this configuration ON-center relay cells receive both excitatory and (indirect) inhibitory feedback from ON-center cortical cells (ON-ON feedback) where the excitatory feedback is fast and spatially narrow while the inhibitory feedback is slow and spatially widespread. In addition to the ON-ON feedback, the connections are accompanied by OFF-ON connections following a so-called phase-reversed (push-pull) arrangement. To facilitate further applications of the model, we have made the Python tool  Visually evoked signals pass the dorsal geniculate nucleus (dLGN) on the route from retina to primary visual cortex in the early visual pathway. This is however not a simple feedforward flow of information, as there is a significant feedback from primary visual cortex back to dLGN. Cortical cells feed back to both relay cells and interneurons in the dLGN, and also to cells in the thalamic reticular nucleus (TRN) which in turn provide feedback to dLGN cells , 2. In tSeveral studies have used computational modeling to investigate cortical feedback effects on spatial and/or temporal visual response properties of dLGN cells –38, 53. extended difference-of-Gaussians (eDOG) model  = xθ(x) is used to enforce non-negative firing rates  − H[−x] = x.With the three assumptions in Eqs In Fourier space we thus haveThe general mathematical expression in K ∼ f(r)h(t)), but space-time coupled kernels can equally be used in the eDOG-model. The same applies to the choice of the receptive-field function of the retinal input ) The mouse seems particularly suitable as model animal since construction and testing of the multiscale models can be greatly facilitated by the ever more sophisticated techniques for controlling gene expression in mice as well as the possibility for optogenetic activation , 116. ThWe envision that at a close and targeted collaboration between modelers and experimentalist, in particular direct assessment of predictions from mechanistic models in targeted experiments, holds great promise for unraveling mechanisms of visual information processing at the different levels of advancements in visual information processing from the earliest sensory systems to more complex computations of the higher cortical areas.S1 FigTop row: predicted area-response curves of relay cells for different arrangements of cortical feedback. Excitatory (left) and inhibitory feedback (center): solid lines and dashed lines correspond to short-delay (5 ms) and long-delay (30 ms) feedback, respectively. Mixed feedback: solid lines correspond to delayed inhibition  are shown as a function of cortical feedback weight for different feedback configurations with different delays. Line styles correspond to different delays as described above. The values on the x-axis represent factors multiplied with the default values for (TIF)Click here for additional data file.


In the present work, the procedures for the creation of self-assembled DNA nanostructures in aqueous and non-aqueous media are described. DNA-Surfactant complex formation renders the DNA soluble in organic solvents offering an exciting way to bridge the transition of DNA origami materials electronics applications. The DNA retains its structural features, and these unique geometries provide an interesting candidate for future electronics and nanofabrication applications with potential for new properties. The DNA architectures were first assembled under aqueous conditions, and then characterized in solution (using circular dichroism (CD) spectroscopy) and on the surface (using atomic force microscopy (AFM)). Following aqueous assembly, the DNA nanostructures were transitioned to a non-aqueous environment, where butanol was chosen for optical compatibility and thermal properties. The retention of DNA hierarchical structure and thermal stability in non-aqueous conditions were confirmed via CD spectroscopy. The formation and characterization of these higher order DNA-surfactant complexes is described in this paper. Rapid development in electronic materials manufacturing has led to the development of standard devices with sub-100 nm features. These advances in science and engineering are remarkable but have reached a fundamental limit using a top-down approach, leading researchers to look towards nature to realize the next level of technological innovation . StructuSpecifically, DNA has been a focus of many studies because it is well-characterized, relatively inexpensive, easy to synthesize, self-sorting, and readily modified with a variety of functional groups. Additionally, DNA is programmable at the molecular level with the ability to pre-organize into well-defined secondary and tertiary structures, and its size is to scale with traditional nanomaterial building blocks . DNA assDespite the promise of DNA assembly, a major hurdle in the use of assembled DNA architectures lies in fact that DNA architectures rely on aqueous phase assembly and are, thereby, not amenable to traditional manufacturing processes . DevelopFor these experiments, a DNA-cationic surfactant complex was created using either hexadecyltrimethylammonium-chloride (CTAC) or bromide (CTAB). This DNA-surfactant complex (DNA-CTMA) rendered the DNA water insoluble, causing it to precipitate out of its aqueous solution, and also had the added benefit of making the DNA more mechanically and thermally stable . The DNAMany different research laboratories have published sequences and protocols for creating a large variety of shapes and structures using differing numbers of DNA strands and different strand lengths . For thiIn order to have a baseline comparison to a non-traditional solvent system, the wwDNA construct was first fabricated in aqueous solution, and characterized using AFM and CD spectroscopy . The wwDAs illustrated in Although the AFM allowed for verification of assembly on the mica surface, the technique does not allow for studying the process in solution. We determined that AFM and CD spectroscopy data could be coupled and used as complementary techniques in order to measure the assembly simultaneously in solution. The wwDNA samples were co-incubated in aqueous solution with freshly cleaved mica, and this mica-DNA solution was monitored via CD and UV spectroscopy and then the mica was imaged via AFM. In order to directly compare results with the published literature, experimental techniques were first validated using a more widely studied DNA system, genomic salmon sperm DNA (saDNA) . The saDThe experimental procedures outlined for the saDNA-CTMA characterization were repeated with the wwDNA-CTMA. As illustrated in Although the spectrum from aqueous to butanol is slightly shifted, this shift is in agreement with literature reports that the overall shape of the spectra remains the same, suggesting retention of overall structure ,32. For In addition to the CD spectroscopy, AFM of the saDNA-CTMA and wwDNA-CTMA was conducted to further confirm sample morphology upon reconstitution with surfactant. These preliminary results are exciting and suggest that, although further optimization is necessary, these techniques should allow for direct utilization of structurally programmable DNA as materials and templates in electronics manufacturing processes.2O). All chemicals and supplies were purchased from Sigma-Aldrich or Invitrogen, and were of the highest grade and purity available. DNA sequences were chosen for their secondary structural characteristics as described in the literature [RP-HPLC purified oligonucleotides were purchased from IDT DNA . DNA samples were reconstituted to a 100 μM final concentration using 1X IDTE (IDT DNA). All aqueous solutions were prepared with nanopure water  and “Wheel 1 comp” (sequence: 5'-CGC TTC GTT TGC GGA ACT GGA GAT GAG CCA TTA CCG AGT AGG TGG ACA GAC C-3').2+ (1 M stock), 2.5 µL Wheel 1 (100 µM stock in TAE), and 2.5 µL Wheel 1 Comp (100 µM stock in TAE), and samples (with and without freshly cleaved mica present) were incubated in a cuvette for CD studies and a microfuge tube for all other experiments. The wagon wheel complex, once formed, is referred to as wwDNA. As seen in The reconstitution of the two single strands of DNA into the double-stranded, tertiary wheel structure was accomplished with the use of a JASCO J-815 CD spectrometer . 1X Tris-Acetate-EDTA (TAE) was diluted from 10X stock solution and combined with 12.5 mM MgCreating the DNA-Surfactant Complex (DNA-CTMA): The process of the DNA-CTMA reaction has been previously studied and explained, and is only being reproduced in this experiment [2O. The centrifugation, supernatant collection, and rinsing steps were repeated two additional times. The samples were placed in an Eppendorf Vacufuge  and lyophilized for about 1 h until samples were dry. The CTAC was chosen over CTAB for future experiments because it is completely soluble at room temperature, whereas the CTAB precipitates at room temperature in aqueous solution and requires an additional heating step.periment . Both CTCD Spectroscopy: The formation of the wagon wheel template in both aqueous and non-aqueous solutions was monitored via CD spectroscopy using a Jasco 850 CD spectrometer , using the temperature interval program. The temperature interval program takes a CD and UV scan at preset temperature intervals (as determined by the user), allowing for direct monitoring of the sample at different temperatures. In addition to direct monitoring of the DNA and DNA-CTMA complex formation, the supernatant of each sample in the DNA-CTMA preparation was analyzed using both CD and UV spectrometry in order to determine the efficiency of the DNA-CTMA complex formation. The absorbance for each sample was tracked to determine the remaining concentrations of DNA left in the supernatant after rinsing the pellet with water.Testing Solubility in Butanol: Lyophilized sample pellets were resuspended in different organic solvents: Isopropyl alcohol, ethanol, methanol, acetonitrile, and butanol [ butanol . ButanolSample Mounting: The samples were mounted on silicon wafers or incubated with freshly cleaved mica in solution. The DNA and DNA-CTMA samples on the silicon wafers were either spin-cast or drop-cast onto the silicon surface. To spin-cast the sample, about 150 µL of the DNA sample was placed onto a silicon wafer and spun in a Laurell Technologies Corporation Spin Processor  at speeds ranging from 1000 to 8000 rpm. For drop-casting, 20–50 µL of the DNA sample was added drop-wise by micropipette onto a silicon wafer and the liquid was allowed to either evaporate under room temperature conditions or samples were incubated for 30 minutes followed by a rinse with ddH2O and dried under N2. Sample uniformity and surface roughness was then characterized by AFM and SEM.Surface Characterization of DNA-Surfactant Complex by Atomic Force Microscopy (AFM): Veeco Nanoman V SPM Atomic Force Microscope , was used in tapping mode with Bruker OTESPA tips  to image the DNA samples.To summarize, these results show for the first time, formation of higher order DNA structures, both in aqueous and non-aqueous solutions. Characterization was performed via AFM (surface) and CD spectroscopy (solution). The aqueous wwDNA samples were combined with surfactant CTAC to form DNA-CTMA complexes, which were soluble in a variety of organic solvents. Future work will include optimization of the material surface for single layer deposition of the templated DNA-CTMA complex, as well as transitioning to a wide range of structures. These nanostructured materials have great promise in creating bottom-up features smaller than those created by current top-down lithographic techniques.


Populus balsamifera, pinpointed 13 genes with differentiated X and Y copies. We tested these genes for differential methylation using whole methylome sequencing of xylem tissue of multiple individuals grown under field conditions in two common gardens. The only SDR gene to show a marked pattern of gender-specific methylation is PbRR9, a member of the two component response regulator (type-A) gene family, involved in cytokinin signalling. It is an ortholog of Arabidopsis genes ARR16 and ARR17. The strongest patterns of differential methylation  are found in the putative promoter and the first intron. The 4th intron is strongly methylated in both sexes and the 5th intron is unmethylated in both sexes. Using a statistical learning algorithm we find that it is possible accurately to assign trees to gender using genome-wide methylation patterns alone. The strongest predictor is the region coincident with PbRR9, showing that this gene stands out against all genes in the genome in having the strongest sex-specific methylation pattern. We propose the hypothesis that PbRR9 has a direct, epigenetically mediated, role in poplar sex determination.Methylation has frequently been implicated in gender determination in plants. The recent discovery of the sex determining region (SDR) of balsam poplar,  Cucumis melo), where an ethylene biosynthesis gene is the key determinantDiospyros and Populus, the determinant of floral sex must become associated with a segregating genomic region, thus becoming a sex locus. In Diospyros a small RNA is a likely candidate for the key determinantPopulus the functional molecular basis has yet to be elucidated.The majority of flowering plants are cosexual (hermaphrodite or monoecious), in marked contrast to animals, which tend to have unisexual individuals. However some 6–7% of flowering plants do have separate sexes (i.e. are dioecious)234Populus (poplars and aspens) methylation has also been implicated in work using the Chinese white poplar (an aspen relative), Populus tomentosa CarrièreSexually specific patterns of methylation have frequently been implicated in plant sex determination8910PopulusP. trichocarpa Torr. & Gray, P. balsamifera L. and P. deltoides W. Bartram ex Marshall, but is different in aspen (P. tremuloides Michx)13The first detailed characterization of the sex-determining region (SDR) in Populus, we wished to examine whole genome methylation patterns of vegetative tissue (xylem) in relation to sex and in particular the methylation status of all the genes identified at the Populus sex-locus by Geraldes et al.To test the hypothesis that sex-specific methylation patterns may be involved in the vegetative-phase establishment of sexual differentiation in Overall we detected expected amounts of DNA methylation across the genome. Taking all the samples , S1 togeet al.Arabidopsis genes ARR16 (AT2G40670) and ARR17 (AT3G56380). All these genes are members of the type-A subfamily, which is generally considered to form part of the network transducing cytokinin signals15We next examined all the genes identified at the sex locus by Geraldes  ARR16 ATG40670 an ARR17 ATG56380. AWe also surveyed sex-specific DNA methylation genome-wide both directly (data not shown) and by using machine learning (see below) to predict gender from genome-wide methylation patterns. We performed this analysis separately for all DNA methylation contexts. Although relatively weak signals of sex-specific methylation are detectable from across the genome (1376 features) the region of the genome coincident with PbRR9 gives by far the most prevalent contribution to the predictive signal , right. Arabidopsis only about 5% of expressed genes are methylated at the proximal promoter20Looking at DNA methylation across the PbRR9 gene, some striking patterns are immediately evident , 2 and 317Knowing the sex of individual poplar trees has allowed us to determine a strong sex bias in methylation at a single locus. Now we ask the question, can knowing the genome-wide pattern of methylation allow us to determine tree gender? Using 70% of the samples as a training set , the metet al.cis or trans or both.As the SDR on chromosome 19 is the only part of the genome that segregates with gender, a gene or genes in this region must ultimately control gender. Geraldes Our study was highly targeted in that it looked for sex-specific DNA methylation in a vegetative tissue (xylem) with no sexual phenotype. This is to test the hypothesis that sex-specific methylation patterns may be involved in the pre-reproductive establishment of sexual development. It would be of interest to examine sex-specific methylation in other tissues including reproductive ones. However, the problem of using reproductive tissue is that they differ in physiology, biochemistry and morphology between sexes, so differences in methylation may be due to the sex of the individual, or merely to tissue differences between the tested organs. Testing a non-sexually differentiated tissue allows determination of constitutive sex differences and establishes that DNA methylation is a marker of sex in tissues that have no phenotypic indications of sex, i.e. the methylome precedes the phenotype as a marker of sex.The sex-specific methylation patterns of PbRR9 could be coincidental or responsible merely for secondary sexual characteristics of little relevance to sex determination. However, this gene has a number of features that make a direct role in sex-determination plausible. Type-A two component response regulators are a moderately-sized family of transcriptional activators with a variety of developmental effects23242526The hypothesis that PbRR9 is directly involved in poplar gender determination clearly deserves to be tested further, especially by detailed expression studies of this gene against the background of reproductive development. A fine-scale study of differential expression against the trajectory of inflorescence development, involving microdissection of inflorescence primordia, would be ideal. It is however, beyond the scope of the present paper. Identifying downstream targets of this gene and assessing the phenotype of plants in which this gene has been misregulated, for instance by VIGS or CRISPR/Cas9 genome editing, would also be obvious possibilities.Silene3132The finding of sex-specific patterns of DNA methylation raises the possibility that inflorescence sex could be altered by hypomethylating chemical treatment, such as by 5-azacytidineP. balsamifera that were collected throughout the natural range of this species. Two common gardens were used to ascertain the effect of between-site differences in environment on the methylation patterns observed. A fully randomised design was used for the common gardens to eliminate any within-site systematic environmental effects on phenotype or epigenome. The collection of P. balsamifera genotypes used here, the AgCanBaP collection of the Agriculture and Agri-Food Canada (AAFC), has been previously describedet al.DNA methylation patterns can vary according to genotype and environmental conditions and so careful replication was used in the experiemntal design, with multiple genetic individuals and multiple sites. The material sequenced consisted of genomic DNA from xylem tissue collected from multiple genotypes in replicate from two environmentally divergent common gardensP. balsamifera (c. 1–2 cm diam.) from the north-exposed side of the tree. The bark was peeled, and the soft tissue inside  was scraped off with a razor blade and immediately stored on dry ice. Tissue was harvested on five consecutive days in the field at the end of July in 2013 . For consistency, tissue was harvested only during the first half of the day (10 am–2 pm). Xylem was chosen as it is a consistent and homogeneous tissue that has little phenotypic plasticity with environment (unlike leaves).Tissue was harvested in the field from small branches of P. balsamifera is the sister species of the fully sequenced P. trichocarpa which has been the subject of extensive variation studies36P. trichocarpa gene annotations are used throughout.et al.Genomic DNA was extracted according to Doyle and DoyleP. trichocarpa reference genome  using the three-letter aligner Bismark v0.12.5in silico bisulfite converted and non-converted genome sequences, and methylation ratios for each cytosine position were determined as #Cs/(#Cs + #Ts). Downstream processing and calculation of differential methylation was done in R for CG, CHG, and CHH contexts separately42Quality and adapter trimmed reads2-penalized negative log-likelihood functionFor statistical modeling of methylation patterns on gender, methylation information was summarized across the genome for tiles (features) of 500 bp length. Filtering for statistical learning included selection of variable features with higher than median inter-quartile range that were detected in 80% of the samples, i.e. features with little variation across samples were excluded. This retained a total of 243197 features for use in supervised classification. Penalized logistic regression (PLR) implemented in program pelora4445How to cite this article: Bräutigam, K. et al. Sexual epigenetics: gender-specific methylation of a gene in the sex determining region of Populus balsamifera. Sci. Rep.7, 45388; doi: 10.1038/srep45388 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


We identified sequence motifs, which enhance or reduce the ability of the Arabidopsis phage-type RNA polymerases RPOTm , RPOTp , and RPOTmp (active in both organelles) to recognize their promoters in vitro with help of a ‘specificity loop’. The importance of this data for the evolution and function of the organellar RNA polymerases is discussed.Arabidopsis RPOTm and RPOTp enzymes. By comparing different truncated and mutagenized promoter constructs, we observed the same minimal promoter sequence supposed to be needed in vivo for transcription initiation. Moreover, we identified elements of core and flanking sequences, which are of critical importance for promoter recognition and activity in vitro. We further intended to reveal why RPOTmp does not efficiently recognize promoters in vitro and if promoter recognition is based on a structurally defined specificity loop of the plant enzymes as described for the yeast and T7 RNAPs. Interestingly, the exchange of only three amino acids within the putative specificity loop of RPOTmp enabled the enzyme for specific promoter transcription in vitro. Thus, also in plant phage-type RNAPs the specificity loop is engaged in promoter recognition. The results are discussed with respect to their relevance for transcription in organello and to the evolution of RPOT enzymes including the divergence of their functions.The single-subunit RNA polymerase (RNAP) of bacteriophage T7 is able to perform all steps of transcription without additional transcription factors. Dicotyledonous plants possess three phage-type RNAPs, RPOTm—the mitochondrial RNAP, RPOTp—the plastidial RNAP, and RPOTmp—an RNAP active in both organelles. RPOTm and RPOTp, like the T7 polymerase, are able to recognize promoters, while RPOTmp displays no significant promoter specificity in vitro. To find out which promoter motifs are crucial for recognition by the polymerases we performed in vitro transcription assays with recombinant The online version of this article (doi:10.1007/s11103-016-0518-z) contains supplementary material, which is available to authorized users. Escherichia coli phage, N4, needs for efficient transcription the E. coli single-stranded DNA binding protein (SSB). SSB presents the hairpin-form promoter to the N4 RNAP for binding and participates in the elongation of transcription by separating the DNA/RNA hybrid from the RNAP elongation complex to enable multiple rounds of transcription  is imported into plastids. The third RPOT gene (RPOTmp) encodes a protein that is dually targeted into both mitochondria and plastids. Consequently, dicotyledonous angiosperms like Arabidopsis possess two mitochondrial  and two plastidial  phage-type RNAPs. The dual-targeted enzyme is not found in monocotyledonous plants and the basic angiosperm, Nuphar advena , plastids use a plastid-encoded plastid RNA polymerase (PEP). PEP is not a single-subunit enzyme but related to the bacterial RNAPs. It is composed of four core subunits  and one of several nuclear-encoded sigma factors. In photosynthetically active plastids, the chloroplasts, several additional protein factors are associated with the holoenzyme and essential for transcription  to result in construct pCold-His-RPOTmp. For mutagenesis, RPOTmp point mutations were introduced by site-directed mutagenesis using the PhusionTM Site-Directed Mutagenesis Kit  according to the manufacturer’s instructions. Used primers are listed in Supplementary Table S3. Recombinant hexahistidine-tagged enzymes were overexpressed in E. coli BL21 Codon Plus RIL  from resulting construct pCold-His-RPOTmp (RHR) in parallel with the wild-type enzymes. Protein expression was induced with a final concentration of 1 mM IPTG for 20–24 h at 15 °C. The purification of histidine-tagged proteins over Ni2+-NTA agarose was performed as described previously  into pKL23 upstream of the two bacterial terminator sequences hisa and thra, which efficiently stop transcription by both RNAPs  or −15 to +4 (pKL23-Patp8-C), respectively, and were tested for in vitro transcription by recombinant RPOTm and RPOTp , are transcribed by RPOTm and RPOTp  involves the formation of hydrogen bonds between amino acids of a C-terminal specificity loop with certain nucleotides of the promoter sequence  for specific transcription from PtrnM-98 as well as from the mutagenized PtrnM-98-tata promoter, which enhanced transcription by RPOTm and RPOTp as described above  and has been described to be recognized by RPOTm in vitro  in many mitochondrial promoters and some NEP promoters of Arabidopsis


The treatment of unresectable locally advanced non-small cell lung cancer (LA-NSCLC) remains a daunting challenge. To date, the best median survival achieved in a randomized prospective bi-modality clinical trial for LA-NSCLC is 28.7 months for patients who received standard chemoradiotherapy on NRG Oncology RTOG 0617 . To imprLA-NSCLC is highly resistant to conventionally fractionated radiation therapy with cooperative group studies showing locoregional failure rates greater than 60% after definitive chemoradiation . This inThe physical properties of heavy-ion therapy also provide dosimetric advantages for LA-NSCLC . With pain situ vaccine responses.In the era of anti-PD-1-/PDL-1-targeted therapies, another important biological advantage of heavy-ion therapy is its potential immunostimulatory effects. While radiation therapy is known to have complex reactions with the immune system and tumor microenvironment, heavy ions have may have unique immune effects that are distinct from photons or proton therapy. Because of their high LET and RBE, preclinical evidence suggests that heavy ions induce non-apoptotic cell death that is independent of the typical p53, bax/bcl, and p21 signal transduction cascades , 15. TheConventionally fractionated radiation therapy as a single modality has dismal cure rates , and theCurrently used fractionation schedules for carbon therapy differ greatly from standard photon techniques. Given differential biological effects on tumors normal tissues as a function of dose per fraction, hypofractionated approaches are commonplace in the practice of heavy-ion therapy. The use of hypofractionated treatment schedules may allow for a greater number of patients to be treated at select centers at a reduced cost. Coupled with the potential for reduced toxicity and improved outcomes, this could make heavy-ion therapy a cost effective treatment, despite the high upfront costs of building such facilities likely on the order of $200–300 million (USD).As the number of heavy-ion therapy centers is expected to increase in the United States in the coming years, there will be opportunity to explore its role for LA-NSCLC. We have outlined rationale for robust exploration of heavy-ion therapy in LA-NSCLC for the purpose of improving what are presently suboptimal outcomes in this population.All authors have contributed to conceptualization and writing of this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Real-time capturing of the myocardial function through CMR tagging is of great clinical importance. In circular tagging, tagline information manifests itself in k-space in an annular sub-region. This k-space characteristic in combination with polar acquisition schemes can be exploited to speed up the imaging. In our previous works, we showed that by selectively acquiring only this portion of k-space through circular sampling, it is feasible to capture tagline information with an acceleration factor of 8 . We furtMR images with circular tag pattern were acquired from phantom as well as a healthy volunteer through a radial k-space sampling scheme on a 1.5T Siemens TIM Avanto scanner. The phantom data was collected with 504 radial spokes to fulfill the Nyquist sampling rate for a 192192 image matrix which prescribes acquisition of 302 projections. The in-vivo data, acquired with 88 views in 11 heartbeats, consists of 19 frames of a mid-ventricular short axis slice. A Hankel-based algorithm referred to as Polar Fourier Transform (PFT) is implemented for reconstruction of the acquired polar data. To study the performance of the method on undersampled datasets, we subsampled the raw datasets to a much lower extent than the Nyquist rate varying from 252 to 18.Phantom data are shown in Figure The PFT method in combination with radial k-space sampling is highly consistent and robust for circular tagging enabling undersampling factors of 16 that can be exploited towards real-time cardiac imaging. In contrast to commonly used reconstruction techniques for radial acquisitions, which su?er from severe streaking artifacts with undersampled projections, the overall appearance of PFT images even for extreme undersampling is of diagnostic quality.