Huntington's disease (HD) is a monogenic neurodegenerative disorder caused by a CAG‐repeat expansion in the Huntingtin gene. Presence of this expansion signifies certainty of disease onset, but only partly explains age at which onset occurs. Genome‐wide association studies have shown that naturally occurring genetic variability influences HD pathogenesis and disease onset. Investigating the influence of biological traits in the normal population, such as variability in white matter properties, on HD pathogenesis could provide a complementary approach to understanding disease modification. We have previously shown that while white matter diffusivity patterns in the left sensorimotor network were similar in controls and HD gene‐carriers, they were more extreme in the HD group. We hypothesized that the influence of natural variation in diffusivity on effects of HD pathogenesis on white matter is not limited to the sensorimotor network but extends to cognitive, limbic, and visual networks. Using tractography, we investigated 32 bilateral pathways within HD‐related networks, including motor, cognitive, and limbic, and examined diffusivity metrics using principal components analysis. We identified three independent patterns of diffusivity common to controls and HD gene‐carriers that predicted HD status. The first pattern involved almost all tracts, the second was limited to sensorimotor tracts, and the third encompassed cognitive network tracts. Each diffusivity pattern was associated with network specific performance. The consistency in diffusivity patterns across both groups coupled with their association with disease status and task performance indicates that naturally‐occurring patterns of diffusivity can become accentuated in the presence of the HD gene mutation to influence clinical brain function. The expansion length is variable and explains much of the heterogeneity in the age at which mutation‐carriers receive a clinical diagnosis of manifest disease  is caused by a CAG‐repeat‐expansion in the Huntingtin gene  and early manifest HD , and visual and subsequently employed principal components analysis (PCA) on diffusivity metrics extracted from these tracts to identify and parsimoniously describe diffusivity patterns in both controls and HD gene‐mutation carriers. First, we examined whether patterns differed between controls, preHD and earlyHD or if there was some consistency within the control and HD populations. Second, we aimed to distinguish diffusivity patterns linked to key contributors to HD pathology, e.g., CAG repeat length, from those that are independent but still influence HD manifestation. Third, we investigated the relative contribution of the three diffusivity measures i.e., RD, AD, and FA to each pattern of diffusivity or principal component. Finally, we examined the relationship between each PC and both cognitive and motor performance as indexed by the global cognitive composite and grip force, respectively. We predicted that tracts associated with networks more directly affected in HD, in particular those connecting striatal regions would likely show more disruption than cortico‐cortical tracts and that the main patterns of diffusivity would discriminate robustly between controls, preHD and earlyHD gene‐mutation carriers. We also predicted that patterns of natural variation would be associated with HD‐related biological variables, including volume changes and CAG‐repeat length extending to structural connections in other brain networks and therefore representing a wider structural HD phenotype.22.1F = 44%; mean age ± SD: 43.1 ± 9.1) carried the HTT gene with a CAG trinucleotide repeat expansion mutation of ≥ 39 but did not have a clinical diagnosis of HD. Thirteen participants  had early motor manifest HD (UHDRS diagnostic confidence level of 3 or 4), and 79 were controls . A global cognitive composite score was derived from nine cognitive tasks that were completed in testing sessions separate to the MRI procedures: Stroop Word Reading test, Symbol Digit Modality Test, Paced Tapping, Circle Tracing (two conditions), Map Search test, Cancelation task, the Spot the Change visual working memory task, Mental Rotation task  as previously described  with eight images with no diffusion weighting (b = 0 s/mm2) (Siemens) and one image with no diffusion weighting (b = 0 s/mm2) (Philips). Acquisition parameters were TE = 88 ms, TR = 13 s and voxel size 2 × 2 × 2 mm (Siemens); TE = 56ms and TR = 11s and voxel size 1.96 × 1.96 × 2.75 mm (Philips). The diffusion data were preprocessed using standard FSL pipelines  and combined to create an improved anatomical scan for DTI data registration. Prior to analysis, we screened each DTI dataset for artifacts, signal drop‐out and motion. Data were then corrected for distortions caused by eddy currents and motion using eddy_correct in FSL, and vector gradient information updated accordingly. The no‐gradient (B0) image (an averaged B0 image was used for the Siemens data) was then skull‐stripped using the Brain Extraction Tool (BET) and manually corrected. For registration purposes, we also applied BET to the structural T1 image. To improve the quality of the brain mask, we combined and dilated a thresholded segmented image with an eroded brain‐extracted T1 mask, which was then applied to the original brain‐extracted T1 image. We then linearly registered the resultant T1 image to the B0 image using FLIRT  versions of the tract‐specific DTI measures. PCA is a method to identify patterns of correlation among multiple measures and is based on the correlations among those measures. To the extent that the first few principal components account for much of the combined variance in a set of variables, those components serve as a useful approximation of the overall correlation patterns. Principal components are described in terms of their correlations with their constituent variables, and the pattern represented by a component can be understood in terms of these correlations. Furthermore, participants’ original data can be transformed to scores on each principal component. We can then analyze a few component scores rather than a large number of original measures, for example, to examine group differences. This reduces multiple comparisons and ideally contributes to insight regarding systematic patterns that may be related to underlying biology and pathology.rrcov , with and without control for age and CAG, to assess relationships between DTI scores and cognitive or motor measures. With the exception noted above, all analyses were performed with SAS/STAT 14.1 .Initially, we observed that separate PCA of the controls and of the combined preHD and earlyHD group yielded very similar results (see Supporting Information Table S2 for further details). Subsequently, the final PCA was performed on all three groups combined. The data were first screened for univariate outliers with the only cases identified from tracts subsequently removed from the analyses due to general QC issues as described above. As a check of stability and potential multivariate outlier influence, PCA was also performed using Hubert's robust PCA , and visual  across all 32 tracts of the combined control and HD gene‐carrier groups. PCA revealed that the first three principal components explained 56.8% of the overall variance in diffusivity  and to the preHD group  while the scores of controls and preHD were similar . Thus, the pattern of diffusivity in PC1 was significantly more pronounced in earlyHD  and also higher than those in preHD, although the difference was not statistically significant . Again, there was no significant difference in scores between controls and preHD . Thus, the pattern of diffusivity in PC2 was significantly more pronounced in earlyHD.PC2 revealed a diffusivity pattern of increased FA and AD focused mainly on sensorimotor tracts Figure ; Table 2p = .0016) and preHD  and there was no significant difference between controls and preHD . Thus the pattern of diffusivity in PC3 was lesser in earlyHD.Finally, PC3 revealed a diffusivity pattern of increased FA and reduced RD in bilateral connections between the DLPFC and caudate and thalamus, respectively Table , while i3.2t = 4.16, p = .0001) and CAG‐repeat length (0.291 (0.08), t = 3.62, p = .0005) but not an age‐by‐CAG interaction. There was a suggestion of a relationship between PC2 and CAG‐repeat length (0.094 (0.06), t = 1.71, p = .092), but no relationship with age; PC3 was predicted by age (0.039 (0.01), t = 2.76, p = .007) but not CAG‐repeat length. For controls, PC3 was significantly predicted by age (–0.027 (0.01), t = 2.52, p = .014), and there was a suggestion of age related difference in PC1 scores (0.02 (0.01), t = 1.86, p = .07). PC2 scores were not predicted by age.As age and CAG‐repeat length interactions influence HD pathogenesis, we used linear regression to test whether age and CAG‐repeat length were associated with our diffusivity patterns. In the combined preHD and early HD group, the diffusivity pattern evident in PC1 was strongly predicted by both age (0.083 (0.02), X2 = 6.90, p = .009), but PC1 (log odds ratio = 0.166 per SD (0.38) X2 = 0.192, p = .66) and PC2 (log odds ratio = 0.448 per SD (0.4) X2 = 1.13, p = .29) were not.Brain volume loss is a hallmark of HD progression and so we assessed whether diffusivity patterns distinguished preHD from earlyHD independently of whole brain, gray matter, white matter, putamen, caudate, and ventricular volumes. A preliminary model selection demonstrated that only the caudate and lateral ventricle volumes acted as joint predictors of HD status. Adding PC scores to that model, the diffusivity pattern of PC3 was also a significant predictor of disease status (log odds ratio = 1.078 per SD (0.410), 3.3PC coefficients represent correlations between diffusivity measures and their contributions to each pattern of diffusivity. We investigated these associations in a subset of representative tracts for each PC, plotting diffusivity metric values against PC scores with individual data points color‐coded for group membership. As discussed earlier, there was considerable overlap in distribution across the groups Figure . For PC13.4Global cognitive composite scores and grip force index (as a marker of motor performance) were correlated with overall PC scores for both controls and a combined HD group Table . The difLooking at specific tracts, there were correlations between increased AD in PMC tracts, and reduced motor performance bilaterally for HD and in the right hemisphere for controls, that were little diminished when controlling for age and CAG repeat length. RD in the DLPFC—caudate tract was positively correlated with the global cognition in both groups. This was attenuated when controlling for age and CAG and was large enough in controls to suggest that changes may be due to ageing. However, many of the attenuated correlations remained significant in HD. There was also a similar correlation for the DLPFC–thalamic tract for controls but not HD.4In this study, we examined the relationships between AD, RD, and FA within white matter tracts in HD gene expansion mutation‐carriers and healthy controls and identified three independent patterns of diffusivity relationships common to both. Absolute diffusivity levels varied between individuals, while the relationship between AD, RD, and FA was maintained potentially representing natural biological variation of white matter microstructure. All three patterns were more pronounced in HD compared to controls; with two closely related to biological factors associated with the HD gene‐mutation. The first described a widespread pattern of increased AD, RD and reduced FA, while the second was restricted to increased AD and FA in sensorimotor network tracts. In the case of the HD group, these patterns were to some extent driven by CAG repeat length, the primary predictor of HD onset, indicating that HD pathogenesis may accentuate pre‐existing diffusivity patterns that are independent of the HD gene mutation and also present in controls. Further, both patterns were linked to widespread HD‐related brain volume loss reflecting key elements of the structural phenotype of HD. The third pattern, consisting mainly of RD changes in tracts associated with cognitive function, however, while associated with age was independent of both HD‐mediated volume loss and CAG repeat length, but nonetheless, a predictor of HD diagnostic status independently contributing to HD clinical manifestations.In this study, we examined diffusivity across 32 white matter tracts from a number of functional networks and identified three independent patterns of AD, RD and FA relationships common to controls, preHD and earlyHD gene‐carriers. Having previously shown that in left‐sided sensorimotor tracts diffusivity naturally varies for both controls and HD gene‐carriers independently i.e., absolute diffusivity levels differs between individuals but the relationship between AD and RD is maintained  and increased axial diffusivity in the sensorimotor network (as seen in PC2) were both closely related to the overall effects of HD pathogenesis, ultimately contributing to the structural phenotype of HD.The third DLPFC‐based diffusivity pattern associated with cognitive function did, however, remain associated with disease status even when controlling for volumetric markers of HD pathology and CAG repeat length. Changes in this pattern may be due to factors that do not cause HD and the diffusivity properties represented may instead independently influence the effects of the HD mutation. As this particular pattern of diffusivity was associated with age for both controls and HD gene‐carriers, changes here may be related to normal aging. In other words, factors associated with aging may influence the diffusivity properties of frontal lobe white matter tracts involved in networks sub‐serving cognition. In healthy individuals, frontal lobe gray and white matter structure and connectivity have been associated with age‐related cognitive performance loss  that may be associated with reduced myelination of the frontal lobe. Given the known benefits of cognitive training on myelination in healthy individuals , similar therapeutic interventions in HD gene‐carriers may counteract the effects of age and thus positively influence HD pathogenesis before disease onset, akin to the disease‐modifying effects of environmental enrichment in HD animal models . Although this pattern of diffusivity correlated with global cognition in controls only, increased RD in the DLPFC‐caudate tract specifically correlated with cognitive performance in both controls and HD. Furthermore, this effect was reduced once correcting for age and CAG suggesting that while influenced by natural variability, the presence of the HD gene mutation has an independent effect that requires further investigation.HD may not, however, affect all aspects of white matter microstructure in equal measure. The first pattern of widespread change was driven mainly by RD suggesting that the HD gene‐mutation may contribute to widespread demyelination ; C Jauffret, D Justo, S Lehericy, K Nigaud, R Valabrègue ; S Klöppel, E Scheller, L Minkova , A Schoonderbeek, E P ‘t Hart ; C Berna, M Desikan, R Ghosh, D Hensman Moss, E Johnson, P McColgan, G Owen, M Papoutsi, A Razi, J Read, ; D Craufurd ; R Reilmann, N Weber ; H Johnson, J D Long, J Mills ; J Stout, I Labuschagne ; G B Landwehrmeyer, I Mayer .Additional Supporting Information may be found online in the supporting information tab for this article.Supporting InformationClick here for additional data file.


Tamarindus indica L. leaves in chronic sodium fluoride poisoning in rats. Tamarindus indica L. seed extract was also reported to have anti-arthritic efficacy by inhibiting cartilage and bone degrading factors. Therefore, an attempt was made to evaluate the effects of ethanolic extract of Tamarindus indica L. leaves in septic arthritis.Our previous study exhibited free radicals scavenging and antioxidant activities of ethanolic and aqueous extracts of − 1 for consecutive 28 days in rabbits. Septic arthritis was induced in rabbits by single intra-articular inoculation of 104 c.f.u. of Staphylococcus aureus to the left stifle joint and was monitored by bacterial colony count, some relevant biochemical parameters and histopathological interpretation of the affected joint. For efficacy evaluation in septic arthritis, linezolid at 75 mg kg− 1 twice daily for 10 days and the ethanolic extract of Tamarindus indica L. at 500 and 1000 mg kg− 1 for consecutive 14 days were administered orally to the rabbits after 48 h of induction of arthritis.The safety study was performed by oral dosing of ethanolic extract of the plant leaves at 2 g kgTamarindus indica L. leaves ethanolic extract, no significant change between days was found for aspertate aminotransferase, alanine transaminase, alkaline phosphatase, blood urea nitrogen and creatinine compared to day 0 values of the same group. The bacterial colony count of synovial fluid following Staphylococcus aureus inoculation to left stifle joint was found to be 1.08 ± 0.47 and 1.19 ± 0.29 c.f.u. mL− 1 in ethanolic extract low dose and high dose groups respectively, on day 2 which was reduced to 0.057 ± 0.036 c.f.u. mL− 1 and nil on day 16. The test extract was also found to markedly reduce simultaneous glucose difference, total protein ratio of serum and synovial fluid, joint radius and joint narrowing.In sub-acute toxicity study of Tamarindus indica L. leaves at 500 mg kg− 1 and 1000 mg kg− 1 produced anti-arthritic effects against S. aureus induced septic arthritis in rabbits. However, the ethanolic extract at 1000 mg kg− 1 orally for consecutive 14 days showed better effects in septic arthritis.Ethanolic extract of  Tamarindus indica L. leaves containing good quantity of vitamin C, α-carotene, minerals like phosphorus, potassium, calcium, magnesium and antimicrobial properties have the potential to be used as possible alternate therapy in septic arthritis. Tamarindus indica L. leaves extract was reported to have antimicrobial activities against both gram negative and gram positive bacteria  .MIC of linezolid had been performed as per protocol using standard strip [Linezolid EzyMICTM Strip (0.016–256 μg mLS. aureus culture (1 mL) possessing 104 c.f.u. (colony forming unit) mL− 1 concentration in the left stifle joint. Arthritis was confirmed by evaluating clinical symptoms like swelling, redness of the particular joint, lameness, restricted movement, restlessness, anorexia and characteristics of synovial fluid in the inoculated joint.The rabbits were inoculated intra-articularly with single dose of 18 h old − 1 orally twice daily for 10 days which was considered as positive control. In addition, Gr-III containing six apparently healthy female rabbits following induction of septic arthritis was given ethanolic extract of Tamarindus indica L. leaves (EETIL) at 500 mg kg− 1 (low dose ethanolic extract of Tamarindus indica L. leaves: LDEETIL) orally once daily for 14 days. Other six apparently healthy female rabbits in Gr-IV after induction of septic arthritis were administered ethanolic extract of Tamarindus indica L. leaves (EETIL) at 1000 mg kg− 1 (high dose ethanolic extract of Tamarindus indica L. leaves: HDEETIL) orally once daily for 14 days. LNZ and EETIL at low and high dose were employed after 48 h of induction of septic arthritis in Gr-II, Gr-III and Gr-IV, respectively. The day 0 values of different parameters for all the groups without any treatment or inoculation were considered as baseline values. All the groups contained six animals for comparative study and to assess statistical significance.A total of 24 rabbits were divided randomly into 4 groups each containing 6 animals. Six apparently healthy female rabbits were utilized for induction of septic arthritis and were considered as negative control (Gr-I). Another six apparently healthy female rabbits (Gr-II) were utilized for induction of septic arthritis and LNZ was administered at 75 mg kgBlood samples were collected from the marginal vein using tuberculin syringe after proper cleaning of the ear with 95% v/v (volume by volume) alcohol and application of local anaesthetic cream. Gentle pressure was applied using sterile cotton gauge to the collection site to stop the bleeding after blood collection.Knee arthrocentesis of the rabbit was performed via a para-patellar approach. The skin was prepared with sterile solution before collection.Biochemical parameters were analysed in both serum and synovial fluid samples. Synovial fluid samples were centrifuged at 3500 rpm (revolutions per minute) for 15 min and supernatants were taken for analysis . Total pBacterial colony count was conducted as per the standard protocol .Lateral and medial joint space width between distal end of femur and proximal end of tibia-fibula were measured by digital radiograph  with the help of a software  on different days in each animal of all the groups before and after induction of septic arthritis as well as during and after the treatment. The angle between tibia-fibula and femur were maintained at a more or less similar degree during radiography.− 1 (standardized veterinary dose) to reduce pain during slaughter for collection of menesci. The menisci were dissected and fixed in 10% formalin for overnight, and were decalcified in 5% trichloroacetic acid for approximately 8 days. The samples were dehydrated in alcohols 70, 80, and 90% for 1 h each and kept in 95% alcohol overnight. Subsequently the menisci samples were immersed in four containers of cent percent alcohol for 1 h each and then processed for paraffin embedding. Sections of 5 μm were made by Olympus CUT 4055 microtome and these were stained with hematoxylin and eosin. Table The animals were euthanized during daytime with intravenous administration of ketamine at 150 mg kgThe various parameters were scored with the assistance of image analysis software LEICA QWIN under LEICA DM 2000 microscope considering the pre-scheduled gradation. The slides were interpreted in one session and in same lighting condition. Cellularity of cartilages was determined by the presence of nuclei per square area of cartilage matrix. A population that was three standard deviations less than the normal control cartilage was defined as acellular cartilage. The percentage loss of matrix was calculated as length of surface with eroded matrix divided by total surface length in a photo-micrograph. Clustering of chondrocytes was estimated as number of clones formed divided by total number of chondrocytes in a particular photo-micrograph. Pannus formation was evaluated by the ratio of the length of joint surface covered by pannus and total length of joint surface in a photo-micrograph.Instead of orthochromasia in Salter’s study, proteoglycans (appeared red) optical density was measured over green background from safranin-o stained field. Two controls were considered for interpretation keeping one as normal cartilage  and another as untreated infection control cartilage . The values were measured in a scale where maximum red value was 255 and minimum red value was zero.The data were expressed as mean and standard error. Student’s t-test was performed to compare the data between different days within the same group of animals.All six animals in each group were included in the analysis of each parameter.Tamarindus indica L. leaves did not show any toxic or adverse effect at 5000 mg kg− 1 following single oral dosing in rabbit.Ethanolic extract of − 1, 1000 mg kg− 1) below 2000 mg kg− 1 were selected for efficacy study.During the sub-acute oral toxicity study, no significant change in AST, ALT, ALP, BUN and creatinine values was recorded on 7th, 14th, 21st and 30th day compared to the values of 0 day of the same group , oxidase (−ve), urease (+ve) and all the isolates produced acid without gas in glucose, sucrose, maltose and mannitol fermentation which are considered as typical characteristics of standard cultures. All the biochemically confirmed cultures produced ‘nuc’ gene in PCR considered as molecular marker for S. aureus.S. aureus isolates were found resistant against ampicillin, methicillin, cefotaxime, ceftizoxime, gentamicin and ampicillin-sulbactum, amoxicillin-clavulinic acid, ticarcillin-clavulanic acid combinations. However, it was sensitive to linezolid.Bacterial colony count, LDH level in synovial fluid and gross and histopathological changes along with observation of clinical signs like lameness, swelling and pain sensation at the inoculated joints confirmed septic arthritis in rabbits.− 1) as compared to day 2 (1.19 ± 0.49 c.f.u. mL− 1) in arthritic control group  and HDEETIL (0.34 ± 0.033 c.f.u. mL− 1) treated group as compared to day 2 of the same groups (Table − 1) as compared to day 2 whereas colony population was nil in LNZ and HDEETIL treated groups on day 16 . On day 16, a significant reduction below 25 mg dL− 1 was observed in rabbits of Gr-II and Gr-IV but the reduction was non-significant (> 25 mg dL− 1) in Gr-I and Gr-III. However in Gr-III, the difference was relatively lower than Gr-I  Table . The ratA marked improvement in joint space  was noticed on day 16 compared to day 0 in all treatment groups Fig. .Fig. 3DAn overall histopathological scoring Table  was perfTamarindus indica L. leaves was recorded to have no adverse effect during sub-acute toxicity study. Therefore, the dose rates of 500 and 1000 mg kg− 1 were used for efficacy study in induced septic arthritis. Induction of septic arthritis was confirmed by histomorphological, biochemical and microbiological parameters. Culture of the collected pus from affected joint showed typical colonies of S. aureus which was further confirmed by PCR. EETIL was found to have a good antimicrobial potential against S. aureus that caused septic arthritis. Synovial LDH activity was observed to be elevated more than 1000 U L− 1 on day 7 in all the groups. In accordance with the present findings, it was also reported [− 1 on day 4 following inoculation of 104 c.f.u. mL− 1 of S. aureus in the left stifle joint of rabbits which indicated significant inflammation in the joint of all the experimental groups. The simultaneous ratio of total protein in serum and synovial fluid in EETIL treated groups approached the ratio of LNZ treated group on day 16. In the presence of bacterial infection, synovial fluid glucose may be at least 25 mg dL− 1 lower than a simultaneous blood glucose [− 1 on day 16 in Gr-II and Gr-IV (Table Tamarindus indica L. leaves have antibacterial effect against S. aureus including methicillin resistant S. aureus [Tamarindus indica L. leaves like saponins, tannins [Tamarindus indica L. leaves as reported earlier might be added advantage to exert anti-arthritic effect of the extract in septic arthritis [The ethanolic extract of reported  that sam. aureus . Septic  tannins , 31 and  tannins . Potent Staphylococcus aureus in rabbits. Septic arthritis generally requires a long term treatment by antibiotic. Therefore, the ethanolic extract of Tamarindus indica L. Leaves at 1000 mg kg− 1 orally for consecutive 14 days may be an alternative option for treatment of septic arthritis not only in animals but also in human beings as it showed significant antibacterial activity against the causative bacteria and clinical improvement of septic arthritis.The present study suggested that both EETIL and LNZ at the proposed dose rates were effective against septic arthritis caused by


Results show that both loaded formulations have a spherical morphology, nanometric size and negative zeta potential. EE% of TQ and Carum loaded niosomes was about 92.32% ± 2.32 and 86.25% ± 1.85, respectively. Both loaded formulations provided a controlled release compared with free TQ. MTT assay showed that loaded niosomes have more anti-cancer activity compared with Free TQ and free Carum against MCF-7 cancer cell line and these results were confirmed by flow cytometric analysis. Cell cycle analysis showed G2/M arrest in TQ, Nio/TQ and Nio/Carum formulations. TQ, Nio/TQ and Nio/Carum decreased the migration of MCF7 cells remarkedly. These results show that the TQ and Carum loaded niosomes are novel carriers with high efficiency for encapsulation of low soluble phytochemicals and also would be favourable systems for breast cancer treatment.Thymoquinone (TQ), a phytochemical compound found in Carum carvil seeds (C. carvil), has a lot of applications in medical especially cancer therapy. However, TQ has a hydrophobic nature, and because of that, its solubility, permeability and its bioavailability in biological mediums are poor. To diminish these drawbacks, we have designed a herbal carrier composed of Ergosterol , Carum carvil extract (Carum) and nonionic surfactants for herbal cancer treatment. C. carvil was extracted and characterized by GC/Mass. Two different formulations containing TQ and Carum were encapsulated into niosomes  and their properties were compared together. Morphology, size, zeta potential, encapsulation efficiency (EE%), profile release rate,  Some traditional cancer therapies such as surgery and chemotherapy have some drawbacks that fail complete treatment of cancer. Also, chemical anticancer drugs can damage healthy cells because of high dose and prolonged usage2.Cancer has become a global deadly disease Such a way that about 7.6 million death reported in 2008 and based on anticipation, maybe it arrive to 13.1 million death in 20303. Doll and Peto were the first to point out an association between phytochemicals and cancer, suggesting that diets rich in vegetables and fruits reduce the risk of certain cancers4. Thomasset et al. (2006), in a review article, discussed dietary polyphenolic phytochemicals as an anticancer agent. They reported that some phytochemicals such as Genistein, Curcumin, Resveratrol and Epigallocatechin gallate (EGCG) have a promising anticancer effect on prostate and breast cancers5. In another review, Young-Joon Surh et al. (2003) described the mechanism and potential applications of dietary constituents on cancer therapy6. Also, Singh et al. (2017) performed Theoretical studies about inhibition activity of the androgen receptor (AR) of four phytochemical agents against prostate cancer. Their finding showed that Isobavachin have the best binding affinity 7. Carum carvil (C. carvil) is one of the best phytochemical compound that has anticancer potential also known as Persian cumin8. Some research showed that Carum carvil has an antioxidant effect that its antioxidant efficiency is better than Vitamin C9. Because of strong antioxidant activity, some researchers mentioned that it maybe has anticancer effect10. The main constituent of C. carvil is Thymoquinone (TQ) that exhibits high antioxidant effects12. TQ was found to be a good inhibitor in some cancer cell such as ovarian adenocarcinoma, uterine sarcoma, prostate cancer cells and cervical Cancer Cells. and hasn’t any toxic effect on non-neoplastic cells13. Rajput et al. (2013) evaluated apoptosis potential of TQ in breast cancer cells and showed that TQ promotes G1 arrest through translation inhibition of cyclin D114. In similar study, Rajput et al. (2013) evaluated the apoptotic effects of TQ and Tamoxifen on XIAP mediated Akt regulation in breast cancer15. Another study that performed by Subhasis et (2012) proved that TQ and Diosgenin have an apoptotic potential in squamous cell carcinoma16. Despite remarkable success of chemoprevention by bioactive plant components in preclinical settings in animal models, the applicability in real life settings still has a long way to go. Some issues associated with these phytochemicals are low water solubility, poor bioavailability, high doses required, diverse genetic background of individuals at risk, etc17. Moreover, unsupervised concurrent administration of natural biomolecules and synthetic drugs may lead to drug failure due to unexpected severe side effects.From the beginning and especially recent years, phytochemicals experienced an increasing demand for treatments of diseases. These compounds are bioactive that found in fruits, seed and other medical plants20. Encapsulation of food molecules into nanocarriers is being thoroughly investigated as a means to circumvent toxicity effects of synthetic drugs23. Indeed, nano-based formulations could enhance the efficiency of bioactive food components by improving their solubility and hence bioavailability. Additional benefits of nanoformulations are targeted delivery, controlled and sustained release, more interaction with selected cells, etc…25.Nanotechnology is a promising field that has a lot of application in medicine. The use of nanotechnology in pharmaceutical filed called nanomedicine26. Generally, cholesterol served as a helper lipid that can diminish interactions of niosomes with proteins of the immune system and increase their stability in biological fluids27. Niosomes have a lot of unique properties versus liposomes such as chemically stability, long storage time, high bioavailability, low toxicity, accessible and low-cost raw materials and spontaneously loading of hydrophobic and hydrophilic agents29. Niosome can increase resistance and also the stability of active plant agent against degradation environment by encapsulating them and thus improving the bioavailability of phytochemical35. Because of hydrocarbon nature of phytochemicals, another critical goal for loading phytochemicals into niosomes is increasing their solubility in aqueous fluids. Rajput et al. (2015) reported novel multilamellar gold niosomes which were loaded with Akt-SiRNA and TQ and this nanoformulated delivery system had a promising activity against tamoxifen-resistant and Akt-overexpressing MCF7 breast cancer cells36. The targeted and controlled release of TQ and siRNA in MCF-7 cells enhanced the concentration of active agents in the active site and hence improved therapeutic efficacy than direct administration of therapeutic agents.Niosomes (Nio) is an interesting type of colloidal nanocarriers that are made of cholesterol and non-ionic surfactants. Non-ionic surfactants have a hydrophilic head connected to a hydrophobic tail which doesn’t have any charge and is relatively non-toxicin vitro cytotoxicity, flow cytometric analysis, DNA fragmentation and cell migration capacity of samples were also studied to investigate the anti-cancer activity. Results showed that this formulation has a good potential for encapsulating phytochemical compounds and also have promising anti-cancer activity. To the best of our knowledge, there is no information about encapsulating Carum into niosome and characterizing its anti-cancer properties.In this study, we evaluated the properties of TQ, TQ loaded niosome (Nio/TQ), Carum and Carum loaded niosome (Nio/Carum) and compared their efficiency on the breast cancer cell. The morphology, size, release behaviour and encapsulation efficiency of prepared niosomes were characterized. The et al. (2011) evaluated the chemical composition of Tunisian Nigella sativa essential oil by GC–EIMS analysis37. They found that The significant components in the plant were p-cymene (49.48%), α-pinene (5.44%), β-pinene (4.31%) and γ-terpinene (3.69%), Whereas TQ represented only (0.79%). The amount of TQ in Carum was satisfactory high to show an anticancer effect against MCF-7 in comparison to standard TQ. Also, there are some other similar phytochemicals in Carum that maybe have the same function as TQ.There are some beneficial compounds in C. carvil seeds, such as alkaloids, protein and essential oils. TQ is the most valuable compound of Carum carvil that about 406 articles focus on it since 1960. The GC-MS analysis of ethanolic extract revealed the presence of 13 components. The specific components of the essential oil and residue were identified by comparing mass spectra fragmentation pattern and retention time with the library values. As shown in Fig. 38. Basiri et al. (2016) prepared α-Tocopherol-loaded niosome and evaluated its release behaviour. They reported that in the higher concentration of α-Tocopherol, the size of niosome formulation increased39.One of the most important factors of a drug delivery system is stability and monodispresity and to prove that we have to characterize formulations by SEM and DLS analysis. SEM image showed that Carum Fig.  and TQ  loaded n40. The size of blank biosome was 122 nm with PDI value of 0.34  evaluated the size of prepared niosomes in the absence/presence of lycopene  and showed that incorporating of lycopene into niosome can increase the size of the resulted formulation41. However, the size of the niosomes is appropriate for tumor specific accumulation via EPR effect42. There is a good agreement size between SEM and DLS for TQ loaded niosome. A lot of authors mentioned that DLS shows hydrodynamic size of particles43. The hydrodynamic size measured by Dynamic Light Scattering (DLS) is defined as “the size of a hypothetical hard sphere that diffuses in the same fashion as that of the particle being measured”. Technically, in a particular suspension, particles are not absolutely spherical. So that, the particle size that calculated by DLS, is apparent size of particles that considered DLS theory and called Hydrodynamic diameter44. Since in Carum there are a lot of phytochemical compounds that can interact with surface of niosomes, we observed a bigger size in DLS than SEM for this formulation. Stability properties of emulsions are directly dependent on the Zeta potential values. Many authors mentioned that a zeta potential near ±30 mV could guarantee a long term stable formulation45. Zeta potentials for Blank niosome, Carum and TQ loaded niosomes were −19 mV, −22 mV and −41 mV, respectively , Carum and TQ loaded Niosomes Fig. . The hom.34 Fig. . Also si.34 Fig. . Mashal ely Fig. . loadingAdditionally, the Carum loaded niosome was kept at 4 °C for over six months, and less than 12 nm size increase was observed suggesting that this formulation have high stability over time (Data not shown).46. Drug Efficiency Encapsulation (EE%) for TQ and Carum was 92.32% ± 2.32 and 86.25% ± 1.85 indicating the successful loading of phytochemicals into Niosomes. Student t-test analysis was performed and showed that there is a significant difference between EE% of TQ and Carum loaded niosomes (p = 0.012). Because of hydrophobic nature of Carum and TQ, they incorporate in the bilayer of niosomes28. In a similar study, Thakkar & Brijesh (2017) synthesed niosomes containing curcumin and primaquine as an anti-malarial combination with a particle size of 220 nm and EE% of 82.12%46.One of the most critical needs of a nanocarrier is its potential to encapsulate drugs. An ideal nanocarrier should load higher content of drugs47. The in vitro release profile of loaded formulations was investigated via dialysis method. The in vitro release behavior of loaded Niosomes and free TQ (TQ that not loaded into niosome) is shown in Fig. et al. (2017) reported that hydrophobic materials that incorporated in bilayer part of niosome have a slower release rate than hydrophilic ones46. In this way, once intravenously administrated, the loaded niosomes could ensure minimal premature release of TQ during the circulation, and thereby greatly reduce required dose for effectiveness. Figure 48.Release rate performance is a required analysis for nanocarriers and give us an insight from the release behaviour of the drug in the body circulationin vitro cytotoxic effect of the niosome formulations were tested using tetrazolium salt on MCF-7 cell line. Figure The in vivo15. In another study, Rajput et al.36 reported TQ at a 40-μL/mL concentration exhibited 80% viability, whereas the same concentration of Nio-Au-TQ significantly reduced cell viability to 40%36. Here, there is a synergic effect that came from better internalizations of nano carrier and a controlled two phase release of TQ.The anti-tumor activity of TQ, Nio/TQ, Carum, Nio/Carum, balnk noisome and untreated samples was performed on MCF-7 cells in 2 µM concentation for 24 h. Figure 15. Rajput et al.14 explored that TQ promotes G1 arrest through translation inhibition of cyclin D1 and induces apoptosis in breast cancer cells. To evaluate whether activity of TQ, Carum and niosome containing these compounds is related to programmed cell death, the percentage of apoptotic and necrotic cells was measured in treated and untreated MCF-7 cells. As depicted in Fig. et al.49 demonstrated that TQ increases the apoptosis rate in both cervical cancer cells dose dependently. In summary, Fig. Cell cycle analysis of all samples showed a G2/M arrest in blank niosome, TQ, Nio/TQ, Carum and Nio/Carum formulations Fig. . Carum din vitro wound (scratch) assays were performed in MCF-7 cells and the wound healing rate was monitored through the complete clousoure of the scratched. As shown in Fig. 49.Migration is one on the important characteristics of cancer cells and promote cancer metastasis. To determine the effect of TQ, Nio/TQ, Carum, Nio/Carum, balnk niosome and untreated samples on migration, in vivo study to better understand the molecular mechanism of these phytochemical carriers for the further development and establishment of Nio/TQ and Nio/Carum as clinical drugs.TQ despite the many advantages in cancer therapy, have a very low solubility in biological fluids that limits its applications. In this study, we introduced an innovative niosome delivery system with ergosterol  in structure, instead of cholesterol which is usual in noisome synthesis, that encapsulated Carum and/or TQ and provided high stability, nanometric size, good encapsulation efficiency. Our findings suggest that Nio/TQ and Nio/Carum markedly inhibited the proliferation of breast cancer cells in comparison with free TQ and/or free Carum. Flow cytometric analysis and DNA fragmentation assay confirmed the apoptotic efficacy of these drug nanoformulated noisome systems. Furthermore, the migration of cancer cells was also suppressed in Nio/TQ and Nio/Carum formulations. This study suggests that encapsulation of low soluble phytochemicals such as TQ and Carum into niosomes can increases their effectiveness and hence can be considered as chemotherapeutic agents against breast cancer. Further studies should be performed to clarify the influence of other factors such as type of intracellular pathways, cellular uptake and Sorbitane monostearate (Span 60), Polyoxyethylene sorbitan monostearate (Tween 60), MTT reagent, Ergosterol, dimethyl sulfoxide (DMSO) and standard Thymoquinone  was obtained from Sigma . Human breast cancer cell line (MCF7) was purchased from the National Cell Bank of Pasteur Institute, Iran. Dead Cell Apoptosis Kit with Annexin V FITC and PI for flow cytometry acquered from BioLegend, USA. SYBR-Safe DNA gel stain for gel electrophoresis was obtained from Invitrogen, USA.50. The GC-mass instrument was a Varian 5300 gas chromatogram with a 2200 mass detector. The used column was a stationary phase HP-5 ms column with specifications of 60-meter length, 0.25 mm diameter and 0.25 µm thickness. Column oven program starts at 60 °C and hold for 5 min, then increased to 270 °C by a rate of 5 °C/min and hold in this temperature for 5 min. The flow rate of He as the carrier gas was kept at 1 ml/min. The temperature of the injection chamber was 260 °C with a split mode of 80:1. The mass spectrum obtained from the sample was compared with the database of the instrument (NIST-MS).C. carvil plant was received from Kerman, Iran. 50 gr of air-dried C. carvil was crushed, and extraction of the ethanolic solution was performed by Soxhlet. The resulted solution was concentrated by rotary evaporator at 180 rpm, 45 °C and two h52. At the first, stock solutions of each niosome component at concantration of 50 mg/mL in choloroform was prepared. Then, Span 60, Tween 60, ergosterol and Carum or TQ at molar ratios of 30:30:30:10 were add to a RB flasks (50 mL). Rotary evaporator was used for cholorophorm evaporation  at reduced pressure, 60 °C for 60 minutes. Resulted lipid film was hydrated with 5 mL of phosphate-buffered saline (PBS) 7.4 solution at 60 °C for 45 minutes. The obtained milky solution containing MLV niosomes was further sonicated for 10 min by Ultrasonic Bath Sonicator  to produce SUVs that have a more homogenoius sizes. For further homogeneous formulation, filtration by membrane filter was performed .Thin film hydration technique or TFH was used for the synthesis of niosome formulationsScanning electron spectroscopy technique (SEM) was used for morphology observation of formulations . For this procedure, each niosome formolation were added to a double-sided carbon tape, positioned on an aluminium stub and vacuum dried. The samples were sputterd by Au at thickness of 200 nm by a Polaron E5100 for 3 minutes and Ar atmosphere. Then, images of each sample were taken at different magnifications.Assessment of some critical parameters of formulations such as the mean hydrodynamic diameter, The particle size distribution, the polydispersity index (PDI) and zeta potential was performed by Dynamic Light Scattering technique or DLS. . The DLS instrument had a power source of 4.0 mW and a He–Ne laser emitting at 633 nm. It was set for backscattering detection at a fixed scattering angle of 90°. Briefly, 2 mL of each Niosome formulation were diluted 1:20 and filtered with 0.45 um membrane filter  to avoid multiple scattering effects of sedimentations. Then, 2 mL of each sample poured in a polystyrene cuvette and analysed at 25 °C in triplicate to yield mean value.EE% of TQ was performed by UV-Visible spectrophotometer . The spectrum was recorded between 750 and 190 nm to determine the characteristic peak of TQ. TQ at wavelenth of 330 nm had a characteristic peak. Calibration curve of TQ in 5, 10, 30, 50, 100 and 150 µg/mL concentration was recorded at 330 nm. For evaluation of EE% of loaded formulations, they centrifuged at 15100 rpm for 30 min and a temperature of 4 °C . After that, supernatant resulted form centrifuge procedure was analyzed at 330 nm and EE% of TQ in the formulation was calculated as follow:First, one mL of each loaded formulation was added to a dialysis bag  and clipped from two ends and placed in a beaker containig 50 mL PBS 7.4 as a release medium. The beaker was stirred at a constant rate of 150 rpm by a magnetic stirrer at 37 °C for eight hours . At each specific time point, One mL of the released content of dialysis bag to the PBS (release medium) was taken for UV-Vis analysis (at a wavelength of 330 nm) and instantly replaced with new PBS buffer. The percentage of released TQ from niosome presented as release profile. Profiles of release rate were drawn by Graph Pad Prism software .2 at 37 °C. The culture medium was refreshed every two days.The MCF7 cells were cultured in RPMI1640 medium  supplemented with fetal bovine serum (FBS) 10% (v/v) and antibiotics (penicillin 0.1 μg/μL and streptomycin 0.1 μg/μL) in a humidified incubator with an environment of 95% air and 5% COIn vitro cell cytotoxic effect (MTT) of TQ, Nio/TQ, Carum extract, Nio/Carum, at the final concentration of 2 µM, and different concentrations of blank niosomes  on MCF-7. Briefly, the MCF-7 cells were seeded into 96-well plates at 7 × 103 cells per well and incubated for 24 h. then, the incubated cells were treated with mentioned compounds and encubated for 24 h. After the incubation time, 100 mL sterile MTT solution (0.5 mg/mL) was added to the wells of plate and incubated again for 4 hr at 37 °C. finally, 100 μL of DMSO solvent was added. In the end, the absorbance was measured at 570 nm53.54. The distribution of cells in various cell-cycle phases was analyzed by flow cytometer  and Flow max 2.3 software.To investigate the effect of TQ, Nio/TQ, Carum, Nio/Carum and blank niosome formulation, MCF7 cells were treated with two μM of compounds at 37 °C for 24 h. After treatment, the cells were trypsinized by Trypsin-EDTA treatment and washed with PBS. Then cells were stained with 50 μg/mL PI solution containing 0.1% Triton X-100 and 0.1% sodium citrateTo study the apoptosis rate, the treated cells were harvested and stained with Annexin V-FITC with PI for 15 min under dark condition, according to the manufacturer’s protocol . The apoptotic cells were then detected on the flow cytometry, and representative histograms of apoptotic versus live cells and necrotic cells were generated.56 with some modifications. Briefly, 2 × 105 cells were treated with TQ, Nio/TQ, Carum, Nio/Carum and blank niosome  for 24 h. Because of the floating apoptosis cells, the media of cultures was collected and centrifuged at 5000 rpm for 5 minutes. The treated and control negative cells were trypsinized, harvested and washed three times with PBS. The cells were then lysed with 500 μL of lysis buffer (20 mM Tris-HCl (pH 8), 10 mM EDTA (pH 8) and 0.5% Triton X-100). After a 30-min incubation on the ice bath, the lysates were centrifuged at 10,000 × g for 20 min. RNase A was added to the supernatant A at the final concentration of 100 μg/mL at 37 °C for one h.For DNA fragmentation assay was performed based previous studiesThe DNA was extracted with a similar volume of phenol/chloroform for 10 min at 25 °C and next centrifuged at 10,000 × g for 5 min. The upper phase was mixed with an equal volume of cold isopropanol and incubated at −20 °C overnight. DNA was separated by centrifugation at 10,000 × g for 10 min. The pellet was air-dried for 10 min at room temperature and dissolved in distilled water. The DNA samples were electrophoresed on a 2% agarose gel containing 100 μL/100 mL SYBR-Safe DNA gel stain  and photographed by an ultraviolet gel documentation system .49.To determine the effect of all tested compounds on MCF7 cell migration, the cells were seeded on 12-well plates in RPMI 1640 growth medium to reach 90–95% confluency. The next day, similar wound area was introduced to monolayer cells by a sterile yellow pipette tip, and TQ, Nio/TQ, Carum, Nio/Carum and blank niosome in the final concentration of 2 µM (in culture medium) were added to each well. The scratched area without any treatment considered as control. The speed of wound closure was assessed by measuring the scratched area at different time intervals . The ImageJ software  was used to quantify the percentage of cell migrationAll experiments were achieved at three times and values are represented as mean ± SD. All data investigated by One-way ANOVA method. Also a p-value < 0.05 was considered as a significant difference.Supplementary


All hospitalized patients should be screened for malnutrition risk. No universal method exists for pediatric patients.kids), applying them to each inpatient aged 1 month to 17 years over a period of five consecutive weekdays in Helsinki University Hospital, Finland.We performed a cross-sectional study comparing three published malnutrition risk screening tools  participated. We found that 6.2% of the children were acutely malnourished and accurately categorized by the three tools. STRONGkids showed the highest specificity (100%) and positive predictive value (36%). Acute malnutrition seemed to be associated with longer hospital stay (p = 0.051).Of the eligible patients, 67%  contains supplementary material, which is available to authorized users. Malnutrition is an underrecognized problem leading to an increased risk for complications, longer hospital stays, and decreased quality of life , 2. Sevekids) [kids methods include a list of high-risk diagnoses. STAMP and PYMS, but not STRONGkids,require anthropometric measurements.Three validated bedside screening methods for malnutrition risk have been developed in recent years: The Screening Tool for the Assessment of Malnutrition in Paediatrics (STAMP) , the Pedkids) . These skids screening tool over PYMS or STAMP due to its higher accuracy. However, a large European multicenter study [The screening tools aim to detect children whose outcome may improve with dietary intervention. The tools classify children as having low, medium, or high risk for malnutrition. Several studies in different countries and settings have compared the applicability of these tools , 6–15. Ser study  and a men = 8) in October to November 2013. Information from the three screening methods was compiled into a set of questions, and the separate risk scores were drawn from the data  standard deviation (SD) scores and weight-to-height ratio –18. ISO-The study protocol was approved by the Ethics Committee of Helsinki University Hospital. All families provided written informed consent.U test and Kruskal-Wallis test for associations between LOS and malnutrition or risk scores. For the ability to detect acute malnutrition, positive and negative predictive values and sensitivity and specificity were calculated. Statistical significance was set at p < 0.05.SPSS software version 24  was used for statistical analysis: Cohen’s kappa for agreement between nutrition risk tools, chi-square and Fisher’s exact tests for differences between categories, and Mann-Whitney Of the 103 eligible children, aged 1 month to 17 years (mean age 6.1 years), 69 (67%) participated: 33 girls and 36 boys (Table kids the least (16%) children as having a high risk for malnutrition  and STRONGon Table . Cohen’sp = 0.497 for differences in the prevalence of malnutrition across the specialties. All three assessment tools classified these acutely malnourished children as having a high nutritional risk.Height and weight measurements were available for 65/69 children (94.2%). Of these, four (6.2%) were acutely malnourished. They were from cardiac, renal, oncologic, and psychiatric specialties; Of the 65 children, 10 (15.4%) had height below − 2 SD, suggestive of chronic malnutrition, although in six the likely explanation was either a syndrome affecting growth or use of corticosteroids. They were categorized as having medium or high risk by all three methods, except for one child, who was categorized as having a low risk by PYMS.p = 0.051). The LOS for those whose height SD was below and above − 2 SD was 14 days (4–121) and 4.5 days [p = 0.090). Age of the child did not differ between malnourished or well-nourished groups or between the risk scores of any of the screening methods.Children with a high risk for malnutrition stayed in hospital longer Table . Median 4 days 4–1 and 4.5p = 0.194). Of those children with high nutritional risk scores with PYMS, 25 were with and 5 without dietetic contact (p = 0.001); with STAMP, 21 were with and 3 without dietetic contact (p < 0.001); and with STRONGkids, 10 were with and 1 without dietetic contact (p = 0.006). High parental concern for the child’s nutrition was associated with acute malnutrition (p = 0.042) as well as with high PYMS (p < 0.001), STAMP (p = 0.005), and STRONGkids (p = 0.003) risk scores.Three of the four acutely malnourished patients had an ongoing contact either prior or during the present admission as well as active follow-up with a dietitian and one had had dietary counseling in the past  was not statistically significant, possibly due to our small sample size. A recent meta-analysis stresses that the observed association between nutritional risk screening and LOS has not been shown to be causal [Poor nutritional status and high nutritional risk scores have been correlated with increased LOS and treatment costs in several studies , 13, 14.e causal .In conclusion, it is important to recognize the children at risk of becoming malnourished. Early intervention preserves quality of life and diminishes costs and length of hospital stay. Bearing in mind the possible pitfalls, universal and easily applicable screening for malnutrition in inpatients is encouraged.Additional file 1:Table S1. Questions in each of the screening methods: the Pediatric Yorkhill Malnutrition Score (PYMS), the Screening Tool for the Assessment of Malnutrition in Paediatrics (STAMP), and the Screening Tool for Risk of Impaired Nutritional Status and Growth (STRONGkids). (DOC 30 kb)


There are large variations in prognosis among hepatocellular carcinoma (HCC) patients undergoing radiofrequency ablation (RFA). However, current staging or scoring systems hardly discriminate the outcome of HCC patients treated with RFA.A total of 757 treatment‐naïve HCC patients undergoing RFA (derivation cohort) were analyzed to establish a nomogram for disease‐free survival (DFS) based on Cox proportional hazard regression model. Accuracy of the nomogram was assessed and compared with conventional staging or scoring systems. Furthermore, external validation was performed in an independent cohort including 208 patients .P < .001). These results were also reproduced in the validation cohort.Tumor size, tumor number, alpha‐fetoprotein, prothrombin induced by vitamin K absence‐II, lymphocyte count, albumin, and presence of ascites were adopted to construct the prognostic nomogram from the derivation cohort. Calibration curves to predict probability of DFS at 3 and 5 years after RFA showed good agreements between the nomogram and actual observations. The concordance index of the present nomogram was 0.759 , which was superior to those of conventional staging or scoring systems (range 0.505‐0.683, all Our simple‐to‐use nomogram optimized for treatment‐naïve HCC patients undergoing RFA provided better prognostic performance than conventional staging or scoring systems. Nevertheless, their performances to predict the prognosis among those treated with RFA have been largely limited, because such systems were originally designed to cover the full spectrum of HCC patients (from very early to far advanced stage cases), rather than “very early or early stage” HCC patients.In this study, we aimed to establish a prognostic nomogram to predict postprocedural outcomes in HCC patients treated with RFA as a first‐line treatment and to externally validate its predictive performance in an independent cohort.22.13/µL; serum aspartate aminotransferase (AST) or alanine aminotransferase (ALT) level < 10 times the upper limit of normal. Exclusion criteria were as follows: the presence of portal or hepatic vein invasion; the presence of extrahepatic spread; Child‐Pugh class C; any other uncontrolled comorbidities or malignant neoplasm; and a prior liver transplant. During the same period, treatment‐naïve patients with HCC who underwent RFA as an initial treatment at Gangnam Severance Hospital, Seoul, Republic of Korea, were considered eligible for the independent validation cohort. The same inclusion and exclusion criteria were applied.Between February 2005 and December 2014, treatment‐naïve patients with HCC who underwent RFA as an initial treatment at Severance Hospital, Seoul, Republic of Korea, were considered eligible for the derivation cohort. Inclusion criteria were as follows: age ≥ 19 years old; at least one unidimensional lesion measurable according to modified Response Evaluation Criteria in Solid Tumors (mRECIST)The study was performed in accordance with the Declaration of Helsinki and Good Clinical Practice and was approved by the institutional review boards (4‐2018‐0969). Informed consent for the invasive procedures was acquired from all patients.2.2The collected clinical data were as follows: patients' baseline clinical features before the primary RFA  and primary tumor factors such as tumor number and tumor size. Neutrophil‐to‐lymphocyte ratio (NLR) was calculated by dividing absolute neutrophil count by lymphocyte count. Albumin‐bilirubin (ALBI) grade was calculated based on a previous report.2.3The diagnosis of HCC was confirmed based on current practice guidelines of the American Association for the Study of Liver Diseases and the European Association for the Study of the Liver.2.4t test  and chi‐squared test . The primary endpoint used for a model development was disease‐free survival (DFS), defined as the time between RFA and recurrence, death from any cause, or the last date of follow‐up. DFS was calculated using Kaplan‐Meier analysis and compared by log‐rank test. To identify the factors associated with DFS in the derivation cohort, Cox‐regression model was used to calculate the hazard ratio (HR) and 95% confidence interval (CI) of each variable on univariate and multivariate analyses. Factors significantly associated with DFS on multivariate analysis were adopted to construct a nomogram and the weighted risk score of each variable in the model was calculated based on β‐regression coefficient by Cox‐regression model.Variables are expressed as the median (interquartile range [IQR]), or number (%) as appropriate. Differences among continuous and categorical variables were examined for statistical significance with Student's The performance of a novel prognostic model to predict the probability of DFS at 6‐60 months after RFA was estimated as integrated area under the curve (iAUC). For calibration, bootstraps with 1000 resamples were used to compare actual survival and predicted survival derived from the prognostic model at 3 and 5 years after RFA. Furthermore, the overall prognostic performance of the prognostic model was assessed using the concordance index (c‐index). The c‐index was compared between our nomogram and other conventional staging or scoring systems. For external validation, similar methods were applied for the independent cohort.P < .05 was considered statistically significant. All statistical analyses were performed using R software .33.1In total, 757 patients were included in the derivation cohort. Baseline clinical characteristics are described in the Table 3.2P < .05 on univariate analysis including etiology, tumor size, tumor number, AFP, PIVKA‐II, neutrophil count, lymphocyte count, anemia, platelet count, bilirubin, albumin, ascites, AST, ALT, and ALP  levels were entered into a multivariate Cox‐regression model. Finally, seven essential variables including tumor size, tumor number, AFP level, PIVKA‐II level, lymphocyte count, albumin level, and presence of ascites were identified as prognostic factors with statistical significance  according to the tertile of the total sum of allocated scores from the prognostic nomogram; the median DFS rates of low‐, intermediate‐, and high‐risk groups were 106.3, 33.6, and 9.3 months, respectively (3.3P > .05). During the median follow‐up of 42.0 months, 112 patients (53.8%) experienced HCC recurrence. DFS at 3 and 5 years were 41.7% and 31.5%, respectively. There was no significant difference in DFS between the derivation and validation cohorts (P = .711 by log‐rank test).A total of 208 patients were included in the validation cohort. As shown in Table P < .001 by log‐rank test, Figure The discriminative ability of the nomogram among the validation cohort was similar in terms of iAUC metrics to predict DFS at 6‐60 months after RFA Figure A, and ag3.4We also investigated whether NLR and ALBI grade rather than simple lymphocyte count or albumin level have discriminative ability in predicting DFS. There was no statistical differences in NLR and ALBI grade between the validation cohort and derivation cohort Table . On univ4Nomograms are widely used as prognostic tools in routine clinical practice since they have the ability to fulfill desire toward personalized medicine through integrating diverse prognostic and determinant variables.Our study has several strengths. First, even though there have been several studies concerning the prognostic model for HCC patients treated with RFA to overcome the disadvantages of conventional staging or scoring systems,When patients were stratified into low‐, intermediate‐, and high‐risk groups, the median DFS rates were 106.3, 33.6, and 9.3 months, respectively. Hence, our proposed nomogram can be utilized in routine clinical practice in establishing optimal surveillance protocols, guiding treatment strategies, identifying potential candidates for effective adjuvant therapy, or designing future clinical trials. For example, local ablation might be preferred to hepatic resection in terms of its less invasiveness for the low‐risk group, whereas hepatic resection might be preferred in terms of better tumor control for intermediate‐ or high‐risk groups. Furthermore, in order to maximize the therapeutic benefit of costly adjuvant immunotherapy and to optimize the number of patients needed to be treated, high‐risk group might be considered reasonable candidates for such adjuvant immunotherapy.Although our study attempted to overcome the shortcomings of previous studies, several unresolved limitations exist. First, this is an observational study which includes single‐ethnic population in a HBV‐endemic geographic area, which is somewhat subject to selection bias. However, to confirm the reliability of our study with robust evidence, we tried to conduct external validation in an independent cohort where the variables were well balanced in comparison to those of the derivation cohort. Furthermore, since there has been no evidence of a relationship between the ethnicity or the etiology and the prognosis of HCC patients treated with RFA,In conclusion, a simple individualized nomogram optimized for treatment‐naïve HCC patients undergoing RFA as a first‐line treatment allowed more delicate prognostification in terms of HCC recurrence, with the appropriate verification in an independent cohort. This novel prognostic calculator can serve as a useful instrument for not only guiding surveillance schedules and treatment strategies but also designing future prospective clinical trials.5The study was performed in accordance with the Declaration of Helsinki and Good Clinical Practice and was approved by the institutional review boards (4‐2018‐0969). Informed consent for the invasive procedures was acquired from all patients.None declared.All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be submitted for publication. Chang Gon Kim and Beom Kyung Kim designed the study. Hyun Woong Lee, Hye Jin Choi, Jung Il Lee, Hye Won Lee, Seung Up Kim, Jun Yong Park, Do Young Kim, Kwang‐Hyub Han, Han Sang Kim, Kwan Sik Lee, Gyoung Min Kim, Man Deuk Kim, Jong Yoon Won, Do Yun Lee, and Beom Kyung Kim were involved in data acquisition. Chang Gon Kim, Kyung Hwan Kim, Seong Jin Choi, Yongun Kim, and Beom Kyung Kim were involved in data analysis. Chang Gon Kim and Beom Kyung Kim wrote the manuscript. Beom Kyung Kim reviewed, revised, and gave final approval of the manuscript. The results presented in this paper have not been published previously in their entirety or in part, except in abstract form. Click here for additional data file. Click here for additional data file.


In humans, satisfying sexual activity within a pair‐bond plays a significant role in relationship quality and maintenance, beyond reproduction. However, the neural and genetic correlates for this basic species‐supporting function, in response to a pair‐bonded partner, are unknown.Oxtr rs53576) and vasopressin‐ (Avpr1a rs3) receptor genotypes with sexual satisfaction and frequency, among a group of individuals in pair‐bonds (M relationship length = 4.1 years). Participants were scanned twice , about 1‐year apart, while viewing face images of their spouse and a familiar, neutral acquaintance.We examined the neural correlates of oxytocin‐ , social bonding , emotion and memory , hormone control ; and somatosensory and self‐other processing . Sexual frequency interactions also showed activations in the SN and paraventricular hypothalamus for Avpr, and the prefrontal cortex for Oxtr.Sex satisfaction scores showed significant interactions with Oxtr and Avpr gene variants may further amplify both basic and complex neural processes for pair‐bond conservation and well‐being.Satisfying sexual activity in pair‐bonds is associated with activation of subcortical structures that support basic motivational and physiological processes; as well as cortical regions that mediate complex thinking, empathy, and self‐other processes highlighting the multifaceted role of sex in pair‐bonds.  The Oxtr rs53576 marker is a single‐nucleotide polymorphism (SNP) of the Oxtr gene that results in individuals having zero, one, or two G‐alleles . Studies have shown that individuals with a greater number of G (vs. A) alleles display greater empathy, altruism, sensitive parenting, and sociality  and arginine‐vasopressin (AVP) in a variety of social behaviors including trust, cooperation, pair‐bonding, reproduction, maternal care, facial recognition, and the regulation of aggression toward strangers  of individuals in a first‐time marriage  in response to images of the spouse . Participants were scanned twice to examine attachment‐related changes for another study, but in the present research, follow‐up scans allowed us to investigate replicated neural effects. We examined correlations of neural responsivity to images of the spouse (vs.theacquaintance) with self‐reports of sexual satisfaction and frequency, and their interactions with 22.1This study was approved by the Human Subjects committees at the University of California, Santa Barbara (UCSB) and Albert Einstein College of Medicine. Participants were recruited by newspapers, internet ads, and flyers seeking, “newlywed and engaged couples.” Eligibility criteria were: marriage to a first‐time spouse, no children, relationship length (<7 years), overall good health, no fMRI contraindications, nonuse of anti‐depressants or excessive medications, and no major surgeries. All participants provided informed consent and received payment.M = 27.2, SD = 3.4), in committed relationships , soon‐to‐be or recently married with a mean annual household income of $60,000 . Two participants had earned a high‐school degree, 10 a college‐degree, and 6 a M.A. or higher. The ethnic composition of the sample was as follows: three Asian‐American, three Hispanic/Latino, and 12 White/European‐American. At Time 2 (T2), 13 (seven women) subjects completed fMRIs .Participants completed fMRIs and surveys at two visits. 1‐year apart. At Time 1 (T1) partcipants were 18 (10 women) healthy, right‐handed individuals, ages 21 to 32 , matched to the Partner by gender, age, and length of time known. The HFN served as a control for facial familiarity for the imaging analyses. Subjects were asked to provide facial photos of the Partner and HFN which were digitized according to standard procedures, and were displayed using Presentation software .Before scanning, all participants were interviewed to determine an appropriate 2.2.1not sexually) while viewing each face image. To reduce carry‐over effects of viewing the face images, stimuli were followed by a countback task, where subjects were asked to mentally count backwards in increments of seven, starting with a random four‐digit number displayed on the screen. Identical photos were used at T1 and T2. Participants were debriefed and provided emotional ratings using a button‐box while still in the scanner, confirming appropriate emotion elicitation corresponding with the target stimulus . Participants were instructed to think about the Partner or HFN (2.2.2http://www.dnagenotek.com). Saliva was analyzed for the Oxtr rs53576 and Avpr1a rs3 genotype variants. Genotyping of the Oxtr rs53576 single nucleotide polymorphism (SNP) was conducted with MassARRAY Compact system genotyping technology (Assays‐by‐Design) on a panel of custom SNP assays designed using RealSNP and MassARRAY Assay Designer (Sequenom Inc). The protocol involved PCR amplification of 10 ng DNA using SNP specific primers followed by a base extension reaction using iPLEX Gold chemistry (Sequenom Inc.). The final base extension products were treated and spotted on a 384‐pad SpectroCHIP using a ChipSpotter LT nanodispenser (Samsung). A MassARRAY Analyzer Compact MALDI‐TOF‐MS was used for the data acquisition process from the SpectroCHIP. The resulting genotypes were called using MassARRAY Typer Analyzer v4.0 (Sequenom Inc.), and the number of G alleles (0–2) was used as a continuous variable in our analyses. For the Avpr1a rs3 polymorphism the number of repeat sequences was identified via fragment analysis. In this technique, a repeat sequence is specified using a sequence‐specific primer congregated to a fluorescent probe and amplified for detection using polymerase chain reaction (PCR). Microsatellite fragment analysis of Avpr1a rs3 was conducted through capillary electrophoresis . For Avpr1a, 6‐FAM and HEX labeled PCR products were mixed and analyzed in multiplex. Samples were denatured at 92°C for 2‐min and then cooled to 4°C on a MJ Research PT‐100 Peltier Thermal Cycler. Samples were transferred to a 96‐well sequencing plate and assigned well coordinates using Applied Biosystems Foundation Data Collection Version 3.0. Electrophoresis of the samples was performed with the Applied Biosystems 3130xl Prism Genetic Analyzer, using dye/filter set, DS‐30. The data were processed and analyzed using Applied Biosystems GeneMapper Software Version 3.7. Control samples were sequenced to determine the repeat‐size based on total fragment length. A left and right offset of 0.4 was used in the bin set parameter of the GeneMapper software to set the limits for acceptable fragment migration for each repeat. The number of repeat sequences was categorized as “long” versus “short” that were coded as the number of long alleles (0–2) as was done in prior research on pro‐social behavior  the Relationship Assessment Scale  responses. A single‐shot echo planar imaging (EPI) sequence that is sensitive to BOLD contrast was used to acquire 37 slices per repetition time , echo time (TE) of 30 ms, flip angle of 90 degrees, field of view (FOV) of 192 mm, and 64 × 64 acquisition matrix. Prior to the acquisition of BOLD responses, a high‐resolution T1‐weighted sagittal sequence image of the whole‐brain was obtained . All pre and postdata processing was conducted with SPM (5 and 12). Functional EPI volumes were realigned to the first volume, smoothed with a Gaussian kernel of 6 mm, and then normalized to the T1.nii image template. Data were smoothed with a 6 mm smoothing kernel as this is a minimum standard and sufficient for subcortical regions  in the general linear model function. The effects of AVPR1a rs3and OXTR rs53576 were tested in separate models because the impact of these polymorphisms on OT and AVP receptor function is still unclear, as OT and AVP sometimes bind to similar receptors making it difficult to discern their distinct function and binding sites , entering the sex satisfaction or frequency ratings, and genotype , with a spatial extent of ≥15 contiguous voxels. Regions were confirmed with the Atlas of the Human Brain ; weekly sexual activity , PLS ; and relationship satisfaction scores on the RAS . Repeated measures t tests showed no significant differences across time  for relationship ratings. Correlations between the PLS and sex satisfaction and frequency were not significant , except for PLS with sex satisfaction at T2 .Participants reported relatively high levels of sexual satisfaction  vs. T1 HFN images , t(17) = 1.40, p > 0.05; and T2 Partner  vs. T2 HFN images , t(12) = 1.76, p > 0.05.Attractiveness ratings of opposite‐sex face images were provided by six coders  recruited for this task, showing adequate inter‐rater reliability . Ratings showed no objective significant differences in facial attractiveness for: T1 Partner , thalamus, cingulate, AG, ventrolateral and dorsomedial PFC, SFG, and premotor areas. T2‐specific results showed positive correlations with sex satisfaction scores in the ventral pallidum (VP), raphe, pons, caudate tail, occipital gyrus, and SII .P Figure a; bilateP Figure b; right P Figure . The lef3.2.4Avpr  and sexual satisfaction (greater) showed positive associations in the right entorhinal area, amygdala; left insula, IFG, orbitofrontal gyrus (OFG), SFG, IPS, parietal area, SMA; and bilateral dorsolateral PFC. T2‐specific interactions of Avpr  with sexual satisfaction scores showed positive associations in the bilateral hippocampus; left amygdala/entorhinal cortex, caudate tail, inferior colliculus, frontal sulcus, and MFG; and the right cingulate/OFG and insula  at both T1 and T2 with sex satisfaction were seen in the left accumbens, VP and hypothalamus  interactions resulted in T1‐specific localizations in the right VTA/SN, entorhinal area/ hippocampus, STG; bilateral posterior cingulate; left putamen, mid‐insula, and SFG. T2‐specific responses were shown in the left VTA/SN, pons/PAG; bilaterally in the GP; and the right amygdala, lingual gyrs, and temporal/auditory cortex  and sex frequency showed significant interaction effects at T1 and T2 in the right SN , caudate, hippocampus, cingulate, IFG, operculum, AG, MTG, STG, and the PFC. , IPS, and SII. At T2, robust correlations were shown for Avpr and sexual frequency in the lateral SN , and the amygdala .T1‐specific neural effects for 3.2.11OXTR  × greater sex frequency/week showed positive associations in the right GP . For Avpr1a rs3 . Scatterplots show the T2 correlations between Avpr1a rs3 and ventral pallidum (VP) response  emotion and memory ; hormone balance , executive/behavioral control (DLFC), and self‐other, reflective, and sensory processing . These data provide robust evidence of the neural correlates of sex satisfaction in pair‐bonds, as our main results were replicated with fMRI scans of newlywed individuals over the first year of marriage.This study was the first to examine the neural correlates of sexual satisfaction and frequency, and their interaction with Oxtr  and Avpr  variants associated with complex social behaviors, including pair‐bonding, and altruism. For humans, however, mating processes appear to be more complex as reflected by activation of cortical networks that support high‐order thinking, planning, and behavioral control; as well as self‐other, and reflective processes. Thus, the present findings are consistent with theoretical models of sexuality in humans that emphasize both conscious, effortful, and deliberate processes; and automatic, unconscious processes  showed significant correlations with neural activity in reward centers, such as the VTA, SN, caudate, and the VP/GP. These regions are rich in binding sites for dopamine, oxytocin, and vasopressin receptors , and they have been shown in over a dozen studies of romantic love  in response to sexual images predicted a stronger desire for partnered sex, 6 months after  alleles display greater empathy, altruism, sensitive parenting, and sociality , as this was also shown in a previous study examining individuals in long‐term marriages of about 20 years on average  may explain the flexibility in mating strategies observed in humans  with respect to satisfying sex acts. However, as with any study, there are limitations. For example, the small sample size  and lack of a control group remain to be addressed in future studies. However, these issues were mitigated, to some extent, by our emphasis of only effects that were replicated at two scans (conducted one‐year apart). Also, this is a major strength as experts of fMRI research advocating for the reproducibility of fMRI results have suggested that smaller, more constrained samples may produce more consistent effects, as larger sample sizes do not always result in consistent replications. Nevertheless, it will be important to replicate these findings in more diverse and larger samples. Also, future studies may wish to use more varied, multi‐item measures of sex satisfaction as well as examining other types of partner stimuli (such as voices or smell). Also they may wish to examine these processes in couples experiencing sexual and/or relationship issues, and with network analysis and PET for more direct evidence of Oxtr's and Avpr's effects. Nevertheless, the present results are novel as they examine basic biological substrates for satisfying sexual activity, a central factor in pair‐bond initiation and maintenance (Donnelly, This is the first study to examine the neural and genetic correlates of sexual satisfaction and frequency among pair‐bonded individuals in response to a partner's face image. We showed that both subcortical and cortical systems are significantly associated with positive sex acts in pair‐boned humans, and to a greater extent for individuals with 5Oxtr and Avpr genotypes associated with complex social and pair‐bonding behaviors. We conclude that beyond reproduction, a satisfying sex life is a healthy and, rewarding attachment variable that may support individual and relationship well‐being; and to a greater extent for individuals with Oxtr and Avpr genotype variants associated with complex social behaviors.Sexuality in humans is complex and it includes a variety of activities that are not obviously and directly relevant to reproductive success. We found that a positively perceived sex life among pair‐bonded individuals recruits a suite of brain regions associated with reward, emotion, attention, memory, physiological homeostasis; and complex cognitive processing suggesting self‐other integration and empathy. Interestingly reward and hormone‐control effects were stronger for individuals with The authors report no competing interests or conflict of interest to report. Click here for additional data file.