One potential drawback of this method are severe photopenic  artifacts surrounding the bladder and the kidneys in the scatter-corrected PET images, which have been reported to occur frequently in clinical practice. The goal of this work was to investigate the occurrence and impact of these artifacts and, secondly, to evaluate variants of the standard scatter correction method with regard to halo-artifact suppression.Combined positron emission tomography (PET) and magnetic resonance imaging (MRI) targeting the prostate-specific membrane antigen (PSMA) with a 68Ga-PSMA-11-PET/MRI study. For each patient, PET raw data were reconstructed employing six different variants of PET scatter correction: absolute scatter scaling, relative scatter scaling, and relative scatter scaling combined with prompt gamma correction, each of which was combined with a maximum scatter fraction (MaxSF) of MaxSF = 75% or MaxSF = 40%. Evaluation of the reconstructed images with regard to halo-artifact suppression was performed both quantitatively using statistical analysis and qualitatively by two independent readers.Experiments using a dedicated pelvis phantom were conducted to investigate whether the halo-artifact is modality-, tracer-, and/or concentration-dependent. Furthermore, 31 patients with history of prostate cancer were selected from an ongoing 68Ga vs. 18F-FDG). Patient- and phantom-based data indicated that halo-artifacts derive from high organ-to-background activity ratios (OBR) between bladder/kidneys and surrounding soft tissue, with a positive correlation between OBR and halo size. Comparing different variants of scatter correction, reducing the maximum scatter fraction from the default value MaxSF = 75% to MaxSF = 40% was found to efficiently suppress halo-artifacts in both phantom and patient data. In 1 of 31 patients, reducing the maximum scatter fraction provided new PET-based information changing the patient’s diagnosis.The phantom experiments did not reveal any modality-dependency (PET/MRI vs. PET/CT) or tracer-dependency (68Ga-PSMA-11-PET/MRI due to 1) the biodistribution of the PSMA-11-tracer resulting in large OBRs for bladder and kidneys and 2) inaccurate scatter correction methods currently used in clinical routine, which tend to overestimate the scatter contribution. If not compensated for, 68Ga-PSMA-11 uptake pathologies may be masked by halo-artifacts leading to false-negative diagnoses. Reducing the maximum scatter fraction was found to efficiently suppress halo-artifacts.Halo-artifacts are particularly observed for  Especially the introduction of Glu-NH-CO-NH-Lys-[68Gallium-(HBED-CC)] (68Ga-PSMA-11)  is considered to have substantially improved detectability and staging of prostate cancer in both PET/CT [5–10] and, more recently, PET/MRI [11–14]. Particularly, 68Ga-PSMA-11-PET/MRI is very promising as both the excellent diagnostic performance of 68Ga-PSMA-11-PET for lymph node metastases  and the high spatial resolution and soft tissue contrast of MRI, beneficial for imaging the pelvis/abdomen, are combined while minimizing radiation exposure compared to PET/CT.Since the prostate-specific membrane antigen (PSMA) shows substantially increased expression in primary and recurrent prostate cancer cells [68Ga-PSMA-11 is excreted by the urine and collected within the urinary system [5], resulting in extreme activity concentration differences between bladder/kidneys and surrounding background. Thus, large organ-to-background activity ratios (OBRs) sometimes result in photopenic artifacts surrounding the kidneys and the bladder in both PET/CT  and, in particular, PET/MRI . These so-called halo-artifacts pose one potential drawback of the clinical use of 68Ga-PSMA-11-PET/MRI for prostate cancer detection and staging. Both primary prostate cancer and local recurrences after radical prostatectomy are typically located very close to the bladder and any photopenic artifact surrounding the bladder could potentially mask findings, falsify uptake values and thus influence the patients’ diagnosis. Additionally, retroperitoneally between the kidneys, the halo may, potentially, mask metastases and thus impair tumor detectability and staging.PSMA is scarcely expressed in abdominal fat and soft tissue while a large proportion of 68Ga-PSMA-PET/MRI are caused by scatter correction . The most widely used scatter correction technique in clinical PET imaging is based on the single scatter simulation (SSS) algorithm , which analytically estimates the scatter contribution from a coarse initial activity image by employing a simplified physical model. SSS explicitly models only single scatter events while multiple scatter and other physical aspects like scatter from activity located outside the field of view (FOV) are neglected. Due to the simplifications and the potentially inaccurate initial activity estimate, SSS can only estimate the shape of the scatter contribution but not its magnitude [19]. The resulting shift of the estimated scatter contribution with respect to the measured emission data requires additional scaling. There are two different methods to perform scatter scaling which are currently available in clinical practice. Relative scaling scales the estimated scatter obtained by SSS to fit to the scatter tails of the measured PET emission data, i.e., to the measured events outside the patient outline, which are assumed to stem from scatter events only. Absolute scaling, on the other hand, also estimates the distribution of unscattered events based on the initial activity estimate and employing a forward model. This allows to relate the SSS-based scatter estimate to the estimated unscattered component, which further allows for an intrinsic scaling of the estimated scatter, without the need to access the emission data [20]. In principle, relative SSS and absolute SSS differ only in the way the SSS scatter estimate is scaled, i.e., only the magnitude of the final scatter, not its shape is different. However, since the scatter estimate is usually refined iteratively, differences in the initial magnitude may also alter the scatter shape throughout the iterative process, such that both magnitude and shape obtained with relative and absolute SSS differ in general,There is evidence that halo-artifacts in 68Ga-PSMA-11 for example, our experience from daily clinical practice indicates that images based on absolute SSS are, however, less prone to halo-artifacts as compared to relative SSS . If, however, the gammas are scattered prior to being detected, their energy may fall within the energy window. In such a case, coincidences between prompt gammas and annihilation photons cannot be distinguished from true coincidences and degrade the image quality if not compensated for [22]. Prompt gamma correction (PGC) has been shown to strongly improve PET quantification using 76Br, 82Rb, 86Y, and other isotopes having a much higher prompt gamma branching fraction than 68Ga [23–25]. More recently, PGC has also been shown to reduce halo-artifacts around the kidneys in PET/CT [15]. Ignoring prompt gammas may result in scatter overestimation when applying relative SSS . However, PGC is currently not employed in routine clinical 68Ga-PET imaging. In a recently published study, Noto et al. showed that PGC only has a limited effect on the appearance of halo-artifacts in 68Ga-PSMA-PET/MRI [18].Furthermore, in 68Ga-PSMA-11-PET/MRI. However, there are two recent publications demonstrating a positive impact of arm truncation correction on the appearance of halo-artifacts . In the present study, we performed phantom experiments using a dedicated pelvis phantom investigating modality-, tracer-, and OBR-dependency of the halo-artifact. Moreover, we retrospectively collected data from 68Ga-PSMA-11-PET/MRI patient scans revealing halo-artifacts, characterized their size in correlation with the OBR, and evaluated several variants of SSS. We performed quantitative and visual assessments and evaluated which of the SSS variants achieves best image quality in terms of halo-artifact suppression.To the best of our knowledge, there is no publication providing a methodological investigation of the halo-artifact in 27]. Comparative phantom measurements were additionally conducted on a clinical TOF PET/CT system  [28].Phantom and patient measurements were conducted with a clinical non time-of-flight (TOF) PET/MRI system  [29] consisting of an 80 mL bladder insert enclosed by an 11 L polymethyl-methacrylate box mimicking the soft tissue background. The maximum outer dimensions of the phantom are about 40 cm within the transaxial plane and about 20 cm in axial direction, such that it can be entirely covered by a single bed position both with the mMR and the mCT. Both bladder and background were filled with deionized water and 0.9% NaCl for all measurements. For PET/MRI, we used a co-registered PET/CT-based attenuation map as basis for attenuation and scatter correction to avoid artifacts caused by the MR-based attenuation map not considering the phantom box. For each individual measurement described in the following, the phantom was placed on the patient bed such that the bladder insert was located in the center of the PET FOV both within the transaxial plane and along the axial direction. Data were acquired for a single bed position. If not state otherwise, 107 counts (prompts after randoms correction).were acquired in sinogram mode.Measurements were performed using a dedicated pelvis phantom  using 3 iterations and 21 subsets and accounting for randoms, scatter, normalization, and attenuation. For the phantom data acquired with the mCT, data were reconstructed twice: without and with TOF information. Transversal image matrix size of the reconstructed volumes was 344 × 344 (2.09 mm × 2.09 mm) for the mMR and 400 × 400 (2.04 mm × 2.04 mm) for the mCT. Slice thickness was 2.03 mm in both cases. All reconstructed volumes were smoothed employing 3D Gaussian post-smoothing with FWHM = 5 mm in all three dimensions.For reconstruction of the acquired PET raw data, the Siemens e7tools offline reconstruction package was used, versions VA20 (mMR) and VG40 (mCT), respectively. Reconstructions were performed employing the ordinary Poisson OSEM (OP-OSEM) algorithm , employing a 2D region-growing-algorithm in transversal, sagittal, and coronal slice orientation in combination with a 3D interpolation. The locations of the seed points were chosen manually to ensure that the majority of consecutive zero-valued voxels were segmented by the algorithm. Relevant segmentation parameters were kept constant for all PET data sets . The OBR was then plotted against the halo size. For the phantom measurements, the data were fitted against a linear function.The halo was defined as volume of consecutive zero-valued voxels being located either around the kidneys or the bladder. For each reconstructed PET image, the halo was semi-automatically segmented with the image processing platform MITK . Evaluating our phantom measurements, we could clearly demonstrate that these so-called halo-artifacts are neither tracer-dependent  nor modality-dependent (neglecting TOF information and impact of improper attenuation maps). Since halo-artifacts were also present when 18F-FDG was used as tracer and since their size and appearance was comparable to the 68Ga-case, prompt gammas could be excluded as being the main reason for halo-artifacts. Nevertheless, prompt gamma correction (PGC) was found to be beneficial in some cases when combined with relative SSS . In addition, patients were scanned up to 3 h p.i. in our study, potentially resulting in very high OBRs, especially around the bladder.Despite tive SSS , Table 240], have been reported in case of inconsistent data, e.g., for incorrect MR-based attenuation maps . However, for the phantom experiments performed in this work, TOF information was not sufficient to entirely suppress halo-artifacts induced by incorrect scatter estimates. For patient data, inaccurate and truncated attenuation maps, the lack of TOF information and larger time differences between tracer injection and patient investigation, potentially resulting in increased OBRs, may be the reason why non-TOF PET/MRI is more prone to halo-artifacts than PET/CT with TOF capability.The phantom experiments also showed comparable halo-artifacts in PET/MRI and PET/CT. Since the impact of an inaccurate MRI-based attenuation map was excluded by using a PET/CT-derived attenuation map of the phantom, only the PET-components of the two modalities were compared. Incorporating TOF information into reconstruction of the PET/CT data resulted in slightly reduced halo-artifacts, indicating that TOF is able to partially compensate for data inconsistencies induced by inaccurate scatter estimation . This ob68Ga-PSMA-11 but not for 18F-FDG most likely lies in the completely different tracer accumulation in human tissue, with68Ga-PSMA-11 being much more specific, resulting in significantly higher OBRs in clinical practice. Both the phantom experiments and the patient data demonstrated a positive correlation between OBR and occurrence and size of the halo-artifact, i.e., increasing OBR results in increasing halo size. These observations suggest asking the patient to void his bladder directly before PET raw data acquisition, possibly under stimulation by diuretic medication [8]. Thereby, OBRs can, potentially, be minimized helping to suppress halo-artifacts, at least around the bladder.The reason why halo-artifacts have been reported for 68Ga-PSMA-PET/MRI . However, both studies show that halo-artifacts may still be present, even when MLAA-based attenuation maps are employed [18] or when patients are scanned with their arms up [26]. We therefore expect that the results for the default MaxSF = 75% will improve significantly in terms of halo-artifact suppression if some truncation correction method, such as MLAA-based attenuation maps [43] or HUGE [44], is applied, especially in combination with relative scatter scaling (tail-fitting technique). Additional benefits with regard to halo-artifacts may be possible when using attenuation maps including bone information, e.g., derived from atlas-based methods [45]. However, the results of the phantom experiments suggest that inaccurate and especially truncated MR-based attenuation maps are not the only reason for halo-artifacts . In addition, For the patient data, we used standard MR-derived attenuation maps, which neglect bone attenuation and often suffer from truncation, since the patients are usually scanned with their arms down as they were in our study. Two recent publications demonstrated that compensating for or avoiding arm truncation significantly improves the appearance of halo-artifacts in 68Ga-PSMA-11-PET/MRI  it remains open, what other causes for halo-artifacts are present. The implemented SSS algorithm does not explicitly model scatter events caused by activity outside the FOV. However, for the phantom experiments, the entire activity was within the FOV, both in transaxial and axial direction. This suggests that out-of-FOV activity is also to be ruled out as remaining main or single reason for halo-artifacts . Our phantom experiments did not provide any insight into the effect of multiple scatter. Including multiple scatter into the scatter estimation model would change both the shape and magnitude of the obtained scatter estimates and potentially improve halo-artifact appearance. Therefore, scatter correction algorithms explicitly treating multiple scatter should be investigated and, if proven valuable, should be made available in clinical practice.Ruling out several potential causes as single or main reason for halo-artifacts in 18F-FDG-PET/MRI) and Abs75 (default for 68Ga-PSMA-11-PET/MRI) do not sufficiently suppress halo-artifacts. Additionally, it was shown that reducing the maximum scatter fraction from MaxSF = 75% to MaxSF = 40% is highly beneficial to significantly reduce halo-artifacts, as visualized in Figs For the phantom and patient data investigated in this study, both quantitative and visual evaluation showed that the default scatter correction variants, i.e., Rel75 . However, such a scaling would apply to all patients, regardless of the initial scatter fraction, whereas reducing MaxSF only changes the magnitude of the scatter if it is above the threshold specified by MaxSF. In other words, adjusting MaxSF is more flexible since it does not result in a downscaling of the scatter for patients with scatter fractions below MaxSF, and thus for cases which most likely do not suffer from halo-artifacts.As stated in the materials and methods section, reducing MaxSF changes the downscaling of the estimated scatter. Similarly, one could use an additional scaling factor 27,31–33], Monte Carlo simulations , and estimations on patient data . The chosen value proved to work well for the phantom data and for all 31 patient data sets presented in this study. However, it should also be obvious that a single value for MaxSF may not reflect large differences in size across patients. For larger patients, higher values of MaxSF may be better suited to correct for halo-artifacts while at the same time minimizing the effect of the modified scatter scaling to other body regions. A similar idea was presented by Rezaei et al. at the last MIC conference, suggesting a plane-dependent scaling of the scatter estimate obtained by SSS, with the scaling parameters being chosen such that the likelihood function is maximized [46].While the results presented in this work clearly demonstrate that reducing MaxSF improves image quality in terms of halo-artifacts and also improves quantification in the affected regions, the optimal choice of MaxSF is not so obvious. Our choice MaxSF = 40% was motivated by previously published studies estimating the scatter fraction based on phantom measurements [68Ga-PSMA-11-PET/MRI are mandatory. Our presented workaround based on a reduced maximum scatter fraction does not entirely solve the problem of halo-artifacts because it does not target the underlying problem of inaccurate scatter estimation. However, it significantly improves the image quality in terms of halo-artifact suppression and thus helps to improve scientific and clinical evaluation. Images reconstructed with a reduced maximum scatter fraction, e.g., MaxSF = 40%, should be made available in clinical practice and considered in cases were halo-artifacts are present, potentially alongside with images corresponding to the default MaxSF = 75%.The clinical examples presented in Figs 68Ga-PSMA-11-PET/MRI is prone to halo-artifacts surrounding the bladder and the kidneys, which are caused by improper scatter correction and appear with increasing OBR over time. Because their appearance may be detrimental to image interpretation and thus patient diagnosis, strategies to avoid halo-artifacts are recommended. Besides striving for low OBRs we recommend reducing the maximum scatter fraction from 75% to 40% for the currently implemented SSS algorithm, which was shown to efficiently suppress halo-artifacts in all investigated cases. This parameter modification may serve as ad-hoc method to enable clinical 68Ga-PSMA-11-PET/MRI in patients where halo-artifacts are present until a more accurate scatter correction technique becomes available in clinical practice.


Intermittent fasting (IF), a periodic energy restriction, has been shown to provide health benefits equivalent to prolonged fasting or caloric restriction. However, our understanding of the underlying mechanisms of IF-mediated metabolic benefits is limited. Here we show that isocaloric IF improves metabolic homeostasis against diet-induced obesity and metabolic dysfunction primarily through adipose thermogenesis in mice. IF-induced metabolic benefits require fasting-mediated increases of vascular endothelial growth factor (VEGF) expression in white adipose tissue (WAT). Furthermore, periodic adipose-VEGF overexpression could recapitulate the metabolic improvement of IF in non-fasted animals. Importantly, fasting and adipose-VEGF induce alternative activation of adipose macrophage, which is critical for thermogenesis. Human adipose gene analysis further revealed a positive correlation of adipose VEGF-M2 macrophage-WAT browning axis. The present study uncovers the molecular mechanism of IF-mediated metabolic benefit and suggests that isocaloric IF can be a preventive and therapeutic approach against obesity and metabolic disorders. Recent studies have discovered that WAT also contributes to whole-body metabolism by regulating thermogenic activity via the browning of WAT, which increases energy expenditure and improves insulin sensitivity3. In this regard, WAT browning has been suggested as a therapeutic approach for obesity and metabolic diseases. A variety of physiological stimuli  and signaling ligands  have been identified for their potential to induce WAT browning4. In addition, recent studies highlight the critical role of the innate immune response with alternative activation of adipose macrophage (M2 macrophage) in the regulation of metabolic function of adipose tissues, including WAT browning5.While fat  is often associated with development of obesity and type 2 diabetes, it is essential for energy homeostasis by storing excess energy and releasing lipids in response to energy deficits9. For example, cold acclimatization10, exercise11, and environmental stimuli9 promote the expression of adipose-VEGF and hence WAT browning. However, the molecular mechanism underlying VEGF-mediated WAT browning remains elusive.We and others have previously demonstrated that vascular endothelial growth factor in adipose tissues (adipose-VEGF) plays a key role in browning of WAT and maintaining the functional integrity of adipose tissues, suggesting the therapeutic potential of VEGF in the treatment of metabolic disorders12. Fasting brings various positive health impacts, suggesting that modulation of fasting period can be used as a therapeutic intervention14. Intermittent fasting (IF), a periodic energy restriction method, has been shown to provide health benefits equivalent to prolonged fasting or caloric restriction (CR)18. The beneficial effects of IF against aging, cancer, cardiovascular diseases, and neurodegenerative diseases have been studied in both animal models and clinical settings21. However, it is unclear whether the metabolic benefits conferred by IF are primarily mediated by reduced food intake23 or attributed to changes in eating pattern. In particular, the 'alternate day fasting' regimen , which is often used in rodent IF models, could result in underfeeding24. In addition, while WAT is one of the most critical organs for fasting physiology, it is not known whether the molecular alteration of WAT after IF is a driver or an outcome of IF-mediated metabolic benefits.Modern lifestyles favor longer periods of daily energy intake and shorter fasting periods. This erratic eating pattern is associated with metabolic disadvantages and contributes to the current global obesity and diabetes epidemicIn this study, we establish a new IF regimen to investigate the impact of IF under isocaloric conditions and demonstrate that IF improves glucose homeostasis and prevents diet-induced metabolic dysfunction without caloric intake reduction. Mechanistically, we found that WAT is pivotal for mediating IF-induced metabolic benefits via browning of WAT through adipose-VEGF-mediated alternative activation of adipose macrophage (M2 macrophage). Our study unveils a novel mechanism by which IF promotes whole-body homeostasis through browning of WAT by VEGF-mediated macrophage switching.19, we developed a new IF regimen comprising 2 day feeding-1 day fasting periods , IF mice showed lower body weight on both ND and HFD  or 45% high-fat diet (HFD). Compared to mice fed  and HFD  and S1E. and HFD . We spec and HFD . However HFD-PF; . These d HFD-PF; . Consist HFD-PF; . Lipid a HFD-PF; . Notably HFD-PF;  and S2F. HFD-PF; . TogetheWe also tested the therapeutic potential of IF on established metabolic abnormalities by performing 2:1 IF on obese mice induced by HFD for 12 weeks 26  and oxidation , as seen in β3-adrenergic receptor (β3-AR) agonist CL-316243- and cold-induced beige adipocytes28. Together, these results suggest that IF promotes a reduction of inflammation and an increase of thermogenic activities in WAT.It has been suggested that IF promotes various health benefits, yet the underlying mechanism is still not fully understood. Given that adipose tissues are pivotal for supplying energy to other tissues during fasting, and were one of the most affected organs/tissues after IF, we performed transcriptome analysis by RNA sequencing (RNA-seq) on PWAT from mice subjected to IF or AL to gain insight into the genome-wide alteration of adipose tissue gene expression by IF. Excluding HFD-AL, the ND-AL, ND-IF and HFD-IF groups clustered together, indicating that even with HFD feeding, IF induced a global change in transcriptome that resembles gene expression profiles of metabolically normal animals . Overall25 . qPCR ve25 . On the (Ebf2)26 . This fiAdrb3  as well as levels of beige/brown adipose markers  were significantly elevated in PWAT of HFD-IF mice, compared to that of HFD-AL mice , revealed that O2 consumption in HFD-IF mice was increased, particularly during the feeding period , without significant changes in physical activities, compared to HFD-AL mice  was not different between groups , TN-HFD-IF mice showed no significant changes of beige/brown adipose marker gene expression in PWAT, compared to TN-HFD-AL mice  compared to that at normal temperature  condition (30 °C), where the thermogenic activation is significantly restricted-AL mice . This superature . Consistondition , suggestondition . Althoug31, we examined changes of the chemokine and adipokine pathways between HFD-AL and HFD-IF. Out of 710 adipose secretory proteins as defined by Lehr et al.30, several potential adipokine genes were markedly upregulated in HFD-IF PWAT, including Vegfa (VEGF), Cfd (adipsin), Nrg4 (neuregulin 4), and Adipoq (adiponectin), whereas Lep (leptin), which positively correlates with adiposity, was downregulated , is a potential modulator of IF.We next aimed to identify a molecular driver of IF-induced metabolic benefits and adipose thermogenesis. As WAT plays a crucial role in whole-body metabolic homeostasis through the production of numerous secretory proteins including adipokinesegulated  and 3B. nscripts . Given tVegfa expression levels in WAT increase progressively with the fasting duration and the increase can be immediately reversed by refeeding , and conducted IF with this loss-of-function mouse model. Intriguingly, IF had no significant effect on WAT weight and adipocyte size in VEGFAdKO mice fed HFD even with decreased body weight  were intermittently subjected to 45% HFD containing doxycycline (HFD-DOX), similar to IF cycle  in PWAT , compared to VEGFCTRL mice  as well as decreased expression of M1 marker genes  using VEGFAdipoq-Tg mice, whose level is comparable to 24-h fasted mice , we identified 24 out of 31 M2-associated genes displaying a positive correlation with VEGFA, 23 out of 30 beige adipocyte genes displaying a positive correlation, and 29 out of 32 M1-associated genes displaying a negative correlation  project genes48 . The corte genes . In cont ITGB751 . After arelation . Togetherelation .14. For example, the 5:2 diet, which involves CR for 2 non-consecutive days a week and unconstrained eating the other 5 days, has become a popular IF regimen and has a potential to be considered for medical interventions13. However, it remains elusive whether the effects upon IF, often tested by alternate day fasting in rodent models19, are attributed to reduced calorie intake and/or modified eating pattern. The present study, using an isocaloric mouse model with a 2:1 IF regimen, demonstrates calorie-independent metabolic benefits of IF. Together with recent important findings on time-restricted feeding and a fasting-mimicking diet (FMD)53, our data support the significance of not only 'what/how much' but also 'when/how often' in energy intake to sustain energy homeostasis and metabolic health. Our 2:1 IF rodent model provides an excellent platform for examining the mechanisms of isocaloric IF to prevent against various obesity-associated metabolic dysfunction.Accumulating evidence suggests that IF provides various biological benefits in animal models and humans14. However, the key organ/tissue driving the IF-induced metabolic benefits is not clearly defined. In the present study, our results posit that adipose tissues are not only an energy reservoir in fasting, but also play a pivotal role in IF-mediated metabolic benefits, particularly by regulating VEGF production. As sufficient vascularization is essential for adipose tissue development and function including cold-, exercise-, or environment-induced adaptive thermogenesis55, the activation of the adipose-VEGF pathway has been suggested as a new therapeutic strategy for the treatment of obesity and diabetes56. We demonstrate in this study that fasting is a physiological means of increasing adipose Vegfa gene expression  with markedly increased adipose vasculature. Our adipose VEGF loss- and gain-of-function mouse models further illustrate that repetition of fasting-induced adipose-VEGF expression underlies IF-mediated metabolic benefits. Interestingly, elevated VEGF expression was mainly noted in IWAT  or BAT of mice subjected to cold exposure57, whereas fasting-mediated augmentation of VEGF expression was greater in PWAT  than in IWAT or BAT. Consistently, the profound weight loss in PWAT seen in our study , subcutaneous WAT, visceral WAT, or BAT have their distinctive physiological and adaptive metabolic properties. This further suggests that combinatory stimuli to activate both subcutaneous and visceral WAT, such as IF and exercise or IF and cold exposure, could generate synergistic metabolic benefits.Both IF and CR impact on nearly all metabolic organs/tissues including hypothalamus, liver, skeletal muscle, and adipose tissuespression , in addiur study  and othe3, and that adipose-VEGF is clearly implicated in this process9. An improved supply of oxygen and nutrient via increased angiogenesis in adipose tissues can account for the necessity of adipose-VEGF in the adaptive responses to physiological stimuli and stress, but may not fully explain the sufficiency of adipose-VEGF in the induction of adipose thermogenesis as seen in several different transgenic mouse models. Of note, it appears that VEGF receptors, such as VEGFR1 (Flt1) and VEGFR2 (Flk1), are not expressed in preadipocytes or mature adipocytes6, leading us to postulate that VEGF acts indirectly in adipose tissue remodeling. Indeed, a recent study has demonstrated that adipose-VEGF induces endothelial PDGF-CC in response to β3-adrenergic activation, thereby stimulating PDGFRα+-progenitor beige adipocytes in WAT58. However, we did not find substantial changes of Pdgfc expression in the WAT of fasted or VEGFAdipoq-Tg mice (data not shown). Importantly, our data and a recent study59 demonstrated that lifestyle interventions, IF, and CR triggered alternative activation (M2) of adipose macrophage, and the type 2 innate immune response that plays a critical role in this adipose thermogenic remodeling5. These immunological changes were also observed in mice with periodic overexpression of adipose-VEGF (driven by Adipoq-Cre and aP2-Cre), similar to a previous study8, but not in the mice lacking IF-mediated WAT browning due to thermoneutrality (TN-HFD-IF) or loss of adipose-VEGF (VEGFAdKO). In addition, both a one-time 24-h fasting and acute adipose-VEGF overexpression without fasting induce alternative activation of adipose macrophage, whereas depletion of macrophage in the presence of adipose-VEGF overexpression abrogated the WAT browning. Together, our results demonstrate that (1) M2 macrophage polarization in IF mice is not simply a consequence of leanness, but a driving force of adipose thermogenesis and metabolic benefits, and (2) adipose-VEGF plays a role in the regulation of M2 macrophage activation as a rapid adaptive response to energy restriction, likely even prior to angiogenesis. Furthermore, (3) the M1/M2 macrophage balance, rather than the number of macrophage, may be critical for adipose thermogenesis, as all fasted mice had reductions in the number of adipose macrophage regardless of conditions.It is now well appreciated that adipose thermogenesis, particularly browning of WAT, improves whole-body metabolism in response to various external and internal stimuli8. What could be the mechanism of this VEGF-mediated M2 transition? Among various upstream signaling pathways implicated in M2 polarization60, we found elevated type2 cytokines by both fasting  and acute adipose-VEGF induction (IL-5), suggesting that adipose-VEGF may act on other upstream immune cells, such as eosinophils, T-helper cell 2 (Th2), or type 2 innate lymphoid cells (ILC2), to promote M2 polarization61. Therefore, future studies delineating a precise role  and its molecular mechanism  of VEGF signaling in adipose immunometabolism should uncover the fundamental mechanism in WAT browning. Furthermore, other adipose factors that we observed to be affected by IF, such as leptin, neuregulin 4, adiponectin and adipsin, could play a role in fasting-mediated immunometabolic responses. This might be particularly expected for leptin, whose expression level is reduced by IF or fasting in WAT and BAT . Leptin modulates a wide range of immune and inflammatory processes, and leptin-deficient mast cells undergo M2 polarization62. Given that leptin could synergistically act with VEGF 63, it will be of interest to examine the implication of leptin in fasting- and VEGF-mediated metabolic benefits. On the other hand, these adipose factors may be involved in the non-thermogenic effects of IF. Although thermoneutrality markedly reduced the metabolic benefits of IF with inhibition of WAT browning, it did not completely eliminate the IF's effects as adipose-derived factors showed similar gene expression (data not shown). As previous studies demonstrate that neuregulin 4 and adipsin are implicated in inhibition of hepatic steatosis32 and improvement of insulin secretion64, respectively, the non-thermogenic benefits of IF may be attributed to these adipose-derived factors or possibly other physiological responses, such as reduced adipose inflammation and increased insulin sensitivity  whether the beneficial effects of IF last after stopping the fasting ; (2) whether there is any potential harm of IF; and (3) whether IF benefits apply to all generations  and disease conditions. Notably, 5 weeks of IF was able to drive beneficial metabolic effects in 8-month-old mice with HFD-induced obesity (unpublished data), further emphasizing the therapeutic potential of IF in age-associated metabolic abnormalities. To further determine the mechanism of IF, it is necessary to test whether fasting (or IF) induces VEGF expression in human adipose tissues, and whether fasting-induced adipose VEGF promotes M2 polarization and WAT browning in humans. Thus, future studies are warranted to examine the adaptive response of human adipose tissues to fasting or IF intervention via thermogenic and even non-thermogenic pathways, in association with the proposed mechanisms such as circadian rhythm69, gut microbiome changes70, and sleep regulation.IF has been practiced in human clinical settings with various fasting regimens, and most studies revealed similar beneficial health effects such as body weight reduction and increased insulin sensitivityIn summary, our data demonstrate that metabolic benefits of IF are largely mediated by adipose thermogenesis without overall caloric reduction. We showed that fasting-induced adipose-VEGF plays a key role in WAT browning through M2 macrophage activation. This finding is further supported by human adipose tissue gene expression analysis illustrating positive correlations between adipose-VEGF expression and both M2 macrophage and WAT browning. Together, the present study illustrates the role of IF-induced adipose-VEGF in remodeling the immunometabolic property of adipose tissue, highlighting the importance of eating pattern and physiological fasting duration to sustain metabolic homeostasis.aP2-Cre, Adipoq-Cre, Ucp1-Cre, Vegfaflox/flox; TetO-Vegf164, Rosa26rtTA and Rosa26mT/mG mice were described previously72. Age-matched littermate controls were used for each experiment.All animal experimental protocols approved by the Animal Care Committee of the Centre of Phenogenomics conformed to the standards of the Canadian Council on Animal Care. No statistical methods were used to predetermine sample size. The investigators were not blinded to allocation during experiments. The colony was housed in a specific pathogen-free (SPF) facility in ventilated cages with controlled environment settings , 12-h light/dark cycles, and free access to water. For the thermoneutrality experiment, mice were housed at 30 °C. C57BL/6J mice were purchased from the Jackson Laboratory. AdKO, and Ucp1-Cre;Rosa26mT/mG mice were randomly divided into two groups: AL and IF groups. Mice were fed either ND  or 45% HFD . Mice in the IF group were subjected to the 2:1 IF regimen, comprising 1 day of fasting followed by 2 days of feeding; the food was removed at 12:00 PM and then provided again the following day (24 h later) at 12:00 PM. Mice in the AL group were handled equivalently. To induce periodic VEGF overexpression in adipose tissues, VEGFCTRL, VEGFaP2-Tg, and VEGFAdipoq-Tg mice were subjected to 45% HFD-DOX (Research Diets) once every 3 days  with post hoc analysis, Student-Newman-Keuls, using Sigma Stat (SPSS) or PRISM 5.0 (GraphPad). Differences with P < 0.05 were considered statistically significant.All results are presented as mean ± SEM. Statistical significance of differences among groups was determined by two-tailed unpaired and paired Student's Other methods are described in K-HK, C-CH, and H-KS conceived, designed, and supervised the research project. K-HK, YHK, JES, JHL, JHM, SK, MSC, K-OD and J-AY performed mouse metabolic experiments. K-HK and YHK analyzed mouse metabolic data. K-HK, YHK, and JHL performed gene expression analysis with assistance from SK and MSC. Histology was conducted by JZ, and analyzed by K-HK, YHK, KF, and FL. JES performed whole-mount adipose tissue staining for blood vessel and macrophage. JGP and SMIH analyzed mouse RNA-seq data. JGP and K-HK performed human gene correlation analysis. AN, AK, and J-RK provided scientific discussion and technical support. K-HK, C-CH, and H-KS wrote the manuscript. All authors discussed the results and commented on the manuscript.The authors declare no competing financial interests.


For the treatment of malignancy, many therapeutic agents, including small molecules, photosensitizers, immunomodulators, proteins and genes, and so forth, have been loaded into nanocarriers for controllable cancer therapy. Among these nanocarriers, polymeric micelles have been considered as one of the most promising nanocarriers, some of which have already been applied in different stages of clinical trials. The successful advantages of polymeric micelles from bench to bedside are due to their special core/shell structures, which can carry specific drugs in certain disease conditions. Particularly, poly(ethylene glycol)–polylactide (PEG–PLA) micelles have been considered as one of the most promising platforms for drug delivery. The PEG shell effectively prevents the adsorption of proteins and phagocytes, thereby evidently extending the blood circulation period. Meanwhile, the hydrophobic PLA core can effectively encapsulate many therapeutic agents. This review summarizes recent advances in PEG–PLA micelles for the treatment of malignancy. In addition, future perspectives for the development of PEG–PLA micelles as drug delivery systems are also presented. Cancer is one of the major health problems that threaten human life. According to the worldwide statistic, there were 14.1 million new tumor incidences and 8.2 million cancer-related deaths in 2012  which are self-assembled by amphiphilic polymers. They consist of two parts: the hydrophobic part on the inside (core) and hydrophilic part on the outside (shell). Therefore, the hydrophobic core can serve as a solubilization depot for agents with poor aqueous solubility. The hydrophilic shell provides advantages including longer blood circulation time and increased stability in the blood. In addition, polymeric micelles can be functionalized with targeting ligands to enhance tumor accumulation. As a result, the role of polymeric micelles in delivery of hydrophobic therapeutic agents for anticancer therapy is promising and opportunistic.S-lactide) (PLLA), poly(D-(+)-R-lactide) (PDLA), and racemic PDLLA. Interestingly, PLLA and PDLA can form stereocomplexes through physical association of PLLA and PDLA chains  (PNIPAAm) -polylactide (PLA) is one of the most prominent amphipathic polymers, therefore it is very suitable for constructing micelles. PLA is a form of biodegradable and biocompatible polyester derived from renewable resources and approved by the Food and Drug Administration (FDA) for clinical use. The hydrophobicity of PLA makes it suitable for the hydrophobic portion of micelles. PLA has three types of stereoisomers: poly(L-(-)-In this review paper, an overview of PEG–PLA-based micelles utilized for the effective delivery of therapeutic agents possessing varying mechanisms for cancer treatment is discussed, as shown in Scheme via the bloodstream and primarily inhibit the rapid replication of tumor cells. Unfortunately, they also inhibit the rapid growth of healthy cells which are crucial to maintaining normal function of the organism, such as hair follicles, bone marrow, and gastrointestinal tract cells  in aqueous solution (280-fold)  effect to solid tumors, particularly to poorly vascularized tumors . The average size of DTX-PM was 58.2 ± 2.3 nm, which was suitable for passive targeting via an EPR effect for anti-cancer drug delivery. More importantly, the DTX-PM significantly induced apoptosis of HSC-3 cancer cells and effectively suppressed the tumor progression in the HSC-3 xenograft model in vivo  DTX-loaded methoxy poly(ethylene glycol)-poly(lactide) micelles. They studied the permeation effect of micelles within both multicellular tumor spheroids and tumor xenografts in mice. The results indicated that small micelles could enhance the tissue penetration however, the blood half-life was short. Micelles of 100–200 nm showed prolonged circulation time compared with that of smaller or larger micelles, but the penetration was limited. Large micelles (>200 nm) had both short half-life and weak penetration ability, leading to limited tumor accumulation -block-poly(acryloyl carbonate)-block-poly (PEG–PAC–PLA) and folate-PEG–PLA (FA-PEG–PLA) block copolymers for receptor-mediated delivery of paclitaxel (PTX). Remarkably, folate-decorated PTX-loaded crosslinked micelles displayed significantly higher toxicity to KB cells than free PTX. This is most likely due to their much more efficient cellular uptake through FA receptor-mediated endocytosis. Flow cytometry studies also showed that folate-decorated FITC-labeled crosslinked micelles were much more efficiently taken up by KB cells than that of controls without folate ligands. All these results indicated that ligand-conjugated interfacially crosslinked PEG–PLA micelles have great potential in targeted cancer therapy  overexpressed on tumor cells. The results showed that OCT-PM-DTX enhanced intracellular delivery efficiency in cells and exhibited higher retardation of tumor growth in vivo  based on a cross-linked framework interspersed with PEG–PLA micelles. After being internalized into tumor cells, the framework of MNC became swollen and disintegrated within the acidic environment of lysosomes via the proton sponge effect of PEI resulting in the release of nanosized micelles for further penetration  and to increase the antitumor efficacy in a multi-drug resistant tumor model  for 2 h than that of nonpretreated ones -N-dodecyl pyrrole) thieno thiadiazole (TPT-TT) and a biodegradable amphiphilic block copolymer, methoxy poly(ethylene glycol)2K-block-poly2K (mPEG2K–PDLLA2K). As a result, TPT-NPs showed high photothermal conversion efficiency, excellent photostability, and heating reproducibility. The photostability of TPT-TT NPs was much better than that of ICG. Besides, TPT-TT NPs exhibited significant photothermal therapeutic effects toward human cervical carcinoma (HeLa) and human liver hepatocellular carcinoma HepG2 cells  has become an effective alternative in cancer therapy because of the advantages over other treatment methods, such as minimal invasiveness, low toxicity, and high specificity to tumor sites  or free radicals. The reactive oxygen species (ROS) can cause significant cellular damage, destruction of tumor blood vessels, and stimulation of antineoplastic immunity  in solution. However, 4% PpIX-conjugated micelles demonstrated better antitumor efficiency in vivo  is a light triggered method for cancer treatment. Due to the minimal aggressiveness and harmlessness to healthy tissue, PDT can avoid the disadvantages of conventional chemotherapeutic agents, such as serious side effects or multidrug resistances. PDT can kill tumor cells mainly through the generation of singlet oxygen  into a cRGD peptide-poly(ethylene glycol)-block-poly(lactic acid) (cRGD-PEG–PLA) and methoxyl poly(ethylene glycol)- block-poly(lactic acid) (mPEG–PLA) nanomicelle. Under the acidic tumor environment (pH 4.5–5.0), the nanoprobe could be activated to produce fluorescence for tumor detection and 1O2 for effective tumor therapy  is a type of peptide that can target the αAlthough there are few related studies, the PEG–PLA-micelle-based PDT represents a new kind of therapeutic method, which may be used in clinics in the future.in vivo, they often suffer from short half-life and ineffectively activate the immune system. Hence, adjuvants are required to elicit effective immune responses against tumor cells , known as a tumor vaccine, and the induction of antigen-specific T cell-mediated immune responses -decorated PEG–PLA-based nanoparticles encapsulated with siPlk1 (ScFv Her2 -NP si Plk1). This ScFv Her2 -NP siRNA could specifically bind to the Her2 antigen overexpressed on the surface of Her2+ breast cancer cells. Therefore, the antitumor efficiency was evidently improved  block copolymers. A specific anti-transferrin receptor antibody, OX26, was then linked onto the surface of the micelles. The results showed that OX26 conjugation visibly increased the uptake efficiency of micelles by target cell lines (C6) and effectively passed through the blood-brain barrier  system to deliver DNA expression vectors to tumor cells. The DNP system was consisted of a DNA expression vector–cationic peptide nanocomplex (NC) surrounded by a PEG–PLA nanoparticle. The results showed that the DNP system could effectively induce apoptosis of tumor cells and possess highly anti-tumor efficacy in vivo -based gene therapeutics have received an increased amount of attention in the last few decades because of their unique advantages for attacking critical cancer hallmarks  composed of monomethoxy poly(ethylene glycol), poly, and polyarginine. This copolymer can further self-assemble into cationic polymeric nanomicelles for in vivo siRNA delivery. The polymeric nanomicelles showed excellent haemocompatibility and higher cell growth inhibition toward EGFR expressed MCF-7 cells. In vivo experiments further proved the effective tumor growth inhibition effect of the polymeric nanomicelles  is a post-transcriptional gene silencing phenomenon. The discovery of RNAi was through the mechanism of small interfering RNA (siRNA). The mechanism is that siRNA can be incorporated into the RNA-induced silencing complex (RISC) and specifically degrades the target messenger mRNA through complementary base pairing, thereby prohibiting the translation into target proteins –block-poly(lactic acid) micelle). When administered the same dosages, the survival time of the intracranial glioblastoma-bearing model mice was significantly longer in the co-delivery (33.5 days) treated group than that of the groups solely treated with CDX-PEG–PLA-PTX (25.5 days), RGD-PEG-PEI/pORF-hTRAIL . This research proved the high efficacy for co-delivery of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and PTX in the intervention of intracranial glioblastoma by employing tumor-targeted gene carrier RGD-PEG-PEI and brain-targeted micelle CDX-PEG–PLA, respectively , a natural polyphenol of vβ3 integrin-targeting peptide (RGD) functionalized polymeric micelles (RFPMs) based on the use of poly(ethylene glycol)-block-poly (PEG–PLA). DOX was conjugated to the PEG–PLA micelle core and combretastatin A4 was physically encapsulated into the micelles (RFPMs-DOX-CA4). The micelles exhibited sequential release kinetics, resulting in sequential killing of endothelial cells and tumor cells in vitro. In B16-F10 tumor-bearing mice, RFPMs-DOX-CA4 showed stronger tumor growth inhibition and significantly higher survival rate compared to other treatment groups  and their ability to achieve multiple modifications  to enhance cancer therapy.b-PLA NPs might interfere with the activation and function of the hypothalamic-pituitary-gonadal (HPG) axis, which might relate to the nanoreprotoxicity of PEG-b-PLA NPs at both the central neuroendocrine and gonadal levels (Rollerova et al., b-PLA NPs also could cause neuroendocrine disrupting effect in the neonatal female rats (Scsukova et al., It is promising to be hopeful about the future of PEG–PLA micelles given their inherent advantages. However, it should be noted that the safety of these novel concepts is a major concern. Rollerova et al. validated that PEG-JW and SL produced the first draft. SC and DL revised the manuscript. DL, YH, and JG proposed the outline of the article and revised the draft before submission. CW revised the draft carefully. In additional all authors provided final approval of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Surgical thrombectomy in the context of acute renal vein thrombosis (RVT) post-transplantation has had limited success, with considerable variation in the surgical techniques used. Unfortunately, it is usually followed by allograft nephrectomy within a few days if rapid allograft recovery does not ensue. We report a case of acute RVT in which nephrectomy was not performed despite a prolonged requirement for dialysis post-thrombectomy, but with recovery of renal function 2 weeks later. We also report the findings of serial MRI with diffusion-weighted imaging (DW-MRI) throughout the patient’s recovery, which provided novel insights into allograft microvascular perfusion changes post-thrombectomy.A 65-year old patient underwent living-unrelated kidney transplantation complicated by acute RVT. Surgical thrombectomy and irrigation led to a delayed, but significant, recovery of renal function. Serial non-contrast DW-MRI scanning was used to non-invasively assess microvascular renal blood flow post-operatively. Unlike standard Doppler ultrasonography, DW-MRI documented reduced microvascular perfusion initially, with gradual but incomplete recovery that mirrored the partial improvement in renal function.Our findings suggest that surgical thrombectomy may be more effective than previously described if followed by careful patient observation. Moreover, diffusion-weighted MRI appears to provide important insights into the pathophysiology of delayed graft function and deserves further investigation. Renal vein thrombosis (RVT) is a dire complication of renal transplantation, leading to delayed graft function (DGF) and even graft failure. Although case series have described incidence rates of only 0.1–6%, RVT accounts for 21–78% of never-functioning allografts , the vasA key consequence of RVT is reduced microvascular perfusion, which is a phenomenon that also occurs in the much more common problem of DGF due to severe ischemia-reperfusion injury . RegardlAdvances in magnetic resonance imaging (MRI) have enabled the non-invasive measurement of microvascular blood flow without the need for gadolinium contrast. One such method, called diffusion-weighted MRI (DW-MRI), measures the movement of water molecules in tissues, and is routinely used to identify microvascular perfusion deficits in the setting of acute brain ischemia . When coA 65-year-old patient with end-stage renal failure on hemodialysis due to recurrent pyelonephritis was admitted to St. Michael’s Hospital for a living unrelated donor kidney transplant see Fig. . Her resThe patient was brought to the recovery room and remained hemodynamically stable, but urine output rapidly decreased over the ensuing 2 h. An ultrasound was performed immediately, which suggested acute RVT see Fig.  – c. TheAt this point, allograft nephrectomy was contemplated, but the kidney was ultimately re-implanted for several reasons: (1) areas of pink, well-perfused parenchyma had been noted upon initial exploration, (2) the majority of the kidney appeared well-perfused after irrigation, (3) good low-pressure flows were noted through the graft, and (4) the allograft was from a living donor. Following re-anastomosis, the allograft renal vein was noted to be patent without compression by the renal artery, and as such the risk for recurrent renal vein thrombosis was felt to be significantly reduced.Post-operatively, the patient continued to receive intravenous unfractionated heparin for 48 h. Due to a drop in hemoglobin from 90 g/L pre-operatively to 58 g/L on post-operative day (POD) 2, intravenous heparin was held. Serial allograft ultrasound scans were performed within the first post-operative week, which revealed no peri-nephric hematoma and normal resistive indices (RIs) , serum creatinine levels, or Doppler ultrasound. Three cross-sectional studies have described DW-MRI findings in patients with DGF, consistently demonstrating reduced perfusion when compared to controls with preserved renal function , 13, 15.In summary, we provide details of an unusual case of delayed renal recovery following successful management of RVT post-living donor kidney transplantation. We also describe the use of serial DW-MRI scans as a novel means to image the impaired microvascular perfusion associated with a rare cause of DGF, and the subsequent increase in microvascular flow as renal function improved. In contrast, standard Doppler ultrasound imaging did not correlate well with either histology or the patient’s clinical presentation. As impairment of microvascular blood flow is a critical contributor to most forms of DGF, future work studying the utility of DW-MRI will be helpful in defining its role in the evaluation of DGF due to RVT and other causes.


It wasshown that the ratios of liposomes exhibiting noticeable phase separationdecrease in the series POPC, DOPC, DPoPC regardless of anyhigh-Tmelt lipid. Substitution of SM for TMCL led to increased lipidphase segregation. Taking into account the fact that the first and second casescorresponded to a reduction in the thickness of the lipid domains enriched inlow- and high-Tmelt lipids, respectively, our findings indicate thatthe phase behavior depends on thickness mismatch between the ordered anddisordered domains. The dipole modifiers, flavonoids and styrylpyridinium dyes,reduced the phase segregation of membranes composed of SM, Chol, and POPC (orDOPC). The other ternary lipid mixtures tested were not affected by theaddition of dipole modifiers. It is suggested that dipole modifiers address thehydrophobic mismatch through fluidization of the ordered and disordereddomains. The ability of a modifier to partition into the membrane and fluidizethe domains was dictated by the hydrophobicity of modifier molecules, theirgeometric shape, and the packing density of domain-forming lipids. Phloretin,RH 421, and RH 237 proved the most potent among all the modifiers examined.In this study we report on experimental observations of giant unilamellarliposomes composed of ternary mixtures of cholesterol (Chol), phospholipidswith relatively low T Depending on the tilt angle of lipid moleculesand the packing of hydrocarbon tails, the solid bilayer is comprised of thefollowing phases: the solid phase , the gel phase, and the ripplephase, which is typical of saturated phosphocholines [os) and liquid states(dl) attributed to lipids with high and low meltingtemperatures, respectively. The presence of sterols, in particular cholesterol,promotes phase segregation and induces the liquid-ordered state(ol). There is evidence that the coefficient oflateral lipid diffusion in the lo- phase is 2–3 timeslower as compared to the dl-areas [dl+ ol) [melt lipids and cholesterol and exist inthe olo-phase. In recent years there has beengrowing interest in lipid rafts due to their important role in proteintrafficking, signaling, immune response, etc. [The lipid bilayer may have a domain structure determined by immiscible lipidphases coexisting in different aggregate states. Single-component lipidbilayers exist in the solid state at temperatures below the melting points(Tcholines . At a po[ld+ lo) . Since nse, etc. -16. Impose, etc. . It ispet al. [et al.[meltlipid components on the phase separation scenario in liposomes packed with Choland SM before and after the introduction of flavonoids or RH dyes. With avariety of phospholipids, POPC, DOPC and DPoPC, we were able to sequentiallychange the disordered lipid phase thickness of a fluid membrane. Lipid mixturescontaining TMCL were also studied for their ability to modify the thickness ofordered lipid domains.Amphiphilic low-molecular-weight compounds, known as dipole modifiers, inparticular some flavonoids, can influence the equilibrium between the phases.Ostroumova et al. reportedet al. . Althoug [et al. examinedMaterialssn-glycero-3-phosphocholine (DPoPC),1,1’,2,2’-tetramyristoyl cardiolipin (TMCL), porcine brainsphingomyelin (SM), cholesterol (Chol) and lissamine rhodamineB-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (Rh-DPPE).The The following compounds were used in the study: sorbitol, phloretin, phlorizin,quercetin, myricetin, and RH 421 ; RH 237 ;1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine 1 (POPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dipalmitoleoyl-Confocal microscopy of giant unilamellar liposomesmelt phospholipid , 40 mol. % high- Tmelt phospholipid (SM or TMCL), and20 mol. % Chol in chloroform (2 mM). The final Rh-DPPE concentration was 1 mol.%. The liposome suspension was aliquoted for storage. An aliquot without adipole modifier was used as control. Test samples contained 400 μM flavonoid  or 10 μMstyrylpyridinium dye (RH 421 or RH 237). Images were acquired with APOoil-immersion objective lens 100.0 × /1.4 HCX PL using a Leica TCS SP5confocal laser scanning microscope . Liposomeswere examined at 25°C. Rh- DPPE emission was excited at 543 nm (ahelium-neon laser). There is evidence that in lipid bilayer systems with phasesegregation, Rh-DPPE shows partitioning preference mainly for the disorderedliquid phase (ld) [lo) and solid ordered phase remain unlabeled [lo-state, while the dye-unlabeled domains of irregular shapewere assigned to the so-state. Each sample was characterizedby the ratio  of homogeneous and heterogonous vesicles:Giant unilamellar liposomes were prepared by the electroformation techniqueusing the Vesicle Prep Pro machine  on glass slides coatedwith titanium and indium oxides  with a surface specific resistivity of 20–30 Ω/sq. Lateral phase segregation wasvisualized by adding the Rh-DPPE fluorescent probe into a three-componentmixture that consisted of 40 mol. % low-Ti is liposome phase separation (homogeneousld-vesicles or liposomes that carry the lo orso-domains); Ni is the vesicle number in asample with a certain phase scenario (from 0 to 50); and N isthe total liposome number in a sample (50 in each system). The pivalues were obtained by averaging values from four independentexperiments. The data for each lipid system were presented in pie charts, alongwith standard deviations for liposomes with assigned phase behavior.where Fig. 1A ( upper panel)shows findings on possible types of phase behavior in unilamellar membranes comprised of SM (40mol. %), Chol (20 mol. %), and low- Tmelt phospholipids  (see melt).Microphotographs with each type of phase segregation scenario are presentedin Fig. 1B.(upper panel). Phase behavior of ternary mixtures containingSM/Chol/ POPC had been previously examined [os), whereas 30 ± 11% SM/ Chol/POPC vesiclesare enriched in liquid-ordered domains with a circular morphology(ol). The remaining liposomes are vesicleshomogeneously labeled with the fluorescent probe(dl), exhibiting no phasesegregation. Fig. 1A(upper panel) demonstrates that substitution ofPOPC for DOPC in the membrane mixture reduces the number of vesicles withos-domains (19 ± 4%) and increases the numberof liposomes with the lo-state (63 ± 10%). When DPoPC wasused, 82 ± 8% vesicles were homogeneously dye-labeled without noticeablephase separation, while the remaining vesicles contained solid domains. It istempting to suggest that visual phase separation decreases in the series POPC,DOPC, DPoPC, and so does the thickness of the disordered phase(Ldd), which includes differentlow-Tmelt phospholipids, whereas the mismatch(Δd) in the hydrophobic bilayer thickness of thecoexisting liquid-ordered and liquid-disordered phases increases [Fig.1 (lower panel), which presents the data onthe phase separation of TMCL membranes , Chol (20 mol. %) andother low-Tmelt phospholipids (40 mol. %)). One can notice thatTMCL-containing liposomes show phase separation regardless of anylow-Tmelt phospholipids (no homogeneously labeled liposome). Thedifferences between the lipid systems are due to the proportion of vesiclescarrying ol- andos-domains. As noted above, DPoPC contributes tothe lowest thickness of the dl-phase among all thephospholipids tested, which corresponds to the highest Δdvalue, and consequently to the highest energy of ordered domainformation. This explains why DPoPC-containing liposomes showed poor phaseseparation (82 ± 7% liposome have ol-domains)versus the POPC-and DOPC-vesicles that formdl-phases with greater thickness and lowerΔd values with phase separation in most liposomes .examined .We founft part) , 23. As meltphospholipids also suggests a role for Δd in regulatingthe lateral heterogeneity of ternary membranemixtures. Figure 1B(lower panel) depicts microphotographs ofTMCL-containing liposomes with low-Tmelt phospholipids.Figure 1Ademonstratesthat SM to TMCL substitution in themembrane mixture leads to enhanced phase separation. In the case of POPC-(left column) and DOPC-containing bilayers (middlecolumn), the proportion of liposomes with so- domainsincreases, whereas DPoPC-bilayers display a statistically significant increasein the numbers of vesicles with ol-domains(right column). This is attributed to the fact that thepresence of TMCL in place of SM lowers the thickness of the ordered phase anddecreases Δd .Taken together, this substitution finally reduces the energy ofordered domain boundary formation. Overall, the findings inFig. 1 allow one to link the lateralheterogeneity of ternary membranes to the mismatch in the membrane thickness ofthe liquid-ordered and liquid-disordered phases: the degree of phase separationis inversely proportional to Δd values.An analysis of phase behavior scenarios involving various high-TFig. 3A (upperpanel). It is clear that the dipole modifiers decrease membrane phaseseparation, which manifests itself as reduced liposome numbers with geldomains. However, upon incorporation of phloretin, quercetin, or myricetin, thedecline in the number of vesicles with os-domainsis accompanied by a corresponding 40–45% increase in the number ofhomogeneously stained liposomes. The presence of phlorizin, RH 421, and RH 237induced a 30–35% increase in the ratio of vesicles withol-domains and a 5–10% increase in the numberof homogeneous liposomes. The elevated liposome concentrations versushomogeneously labeled DOPC-liposomes in the presence of phloretin, phlorizin,RH 42,1 and RH 237 (by 10–30%) and elimination of vesicles withos-domains in the presence of phloretin could beexplained by decreased phase separation following the addition of dipolemodifiers as in the case with POPC.Figure 3B showsmicrophotographs of lipidvesicles containing DOPC, Chol, and SM and their phase behaviors in the presence of phloretin and RH 421. Nostatistical significance was found regarding the effects of quercetin andmyricetin on phase separation in DOPC membranes.Taking into account the fact that dipole modifiers impact not only the dipolepotential, but also the packing of lipid components -27, we sdvalues under the influence of the agents tested. The most likelyscenario is that the polar heads of lipids take over more space in the membranein response to burying of the modifiers into the lipid layer and dipole-dipoleinteractions between them. As shown by differential scanning calorimetry, thisrelatively increases the mobility of carbohydrate chains and reduces theTmelt of lipids [Changes in the phase separation scenario of SM-containing membranes in thepresence of dipole modifiers could be caused by elevated Δf lipids , 25, 28.f lipids .dl-domainsbecome sensitive to fluidization as compared to ordered domains, sincepartitioning of modifiers into the ld-domains seems to beimpeded in the context of tightly packed lipids. This scenario is schematicallyillustrated in Fig. 2 (left part).In addition to the modifier type, the geometric characteristics of lipidmolecules that form the phase into which a modifier partitions also play aregulating role. In the case of lipids with a cylindrical geometry, such asDOPC, POPC, and SM -31, ld-dFig. 3A,DPoPC-containing membranes exhibited no statistically significant differencesbetween phase behavior scenarios before and after the modifiers had been added.Bearing in mind that no phase separation is observed in most DPoPC-vesicleseven in the absence of dipole modifiers due to the greatest mismatch in themembrane thickness of the liquid-ordered and liquid-disordered phases, furtherelevation of Δd does not lead to significant changes inbilayer phase separation.As shown by the lower panel in dl-domainsand ordered domains will not dramatically alter the thickness mismatch betweenthe phases, thus preventing changes in phase behavior scenarios.Figure 4A shows thatregardless of the type of low-Tmelt lipidwithin the model membranes, the presence of a dipole modifier neithersignificantly increases the relative number of TMCL-containing liposomes witho-ldomains nor induces the emergence of liposomeswith noticeable phaseseparation. Fig. 4B showsmi crophotographs of lipid vesicles incorporating DOPC, Chol, and TMCL andliposomes modified with phloretin or RH 421.In contrast to SM, TMCL has an inverted cone shape that triggers invertedspontaneous curvatures of the monolayers formed by it . It is hIn conclusion, our findings suggest a key role for the mismatch thicknessbetween the ordered and disordered phases in modulating phase behaviorscenarios in ternary model membranes. It is believed that our work will open upnew a venues for research into the use of dipole modifiers for the regulationof lipid lateral heterogeneity in bilayers.


The androgen receptor (AR) plays important roles in prostate cancer development and prostate tumor growth. After binding to androgens, AR functions as a nuclear receptor and translocates to the nucleus to bind to specific AR-binding sites (ARBSs). AR regulates epigenetic factor recruitments to activate its downstream signaling. Although androgen deprivation therapy (ADT) is initially useful for prostate cancer patients, most patients eventually show resistance with hormone-refractory prostate cancers (HRPCs) or castration-resistant prostate cancers (CRPCs). Thus, new therapeutic strategies targeting HRPCs/CRPCs should be very important for clinical medicine as well as prostate cancer biology. Past studies have shown that mechanisms such as AR overexpression, hypersensitivity, variants and reprograming are responsible for developing HRPCs/CRPCs. These findings suggest that AR target genes will be major key factors. In this review article, we focus mainly on the androgen-regulated microRNAs (miRNAs) to summarize the contribution of miRNA-mediated pathways for prostate cancer progression. Non-coding RNAs (ncRNAs) are RNA transcripts that do not encode for proteins and can be divided into two major groups: small ncRNAs, between 18 and 200 nucleotides (nt) in length, and long non-coding RNAs (lncRNAs) which are larger than 200 nt. Among small ncRNAs, microRNAs (miRNAs) are evolutionally conserved single-stranded small non-protein coding transcripts of approximately 18–22 nt that post-transcriptionally regulate gene expression . SeveralProstate cancer is one of the leading causes of cancer morbidity and mortality in developed countries. Androgen receptor (AR) and its downstream signaling are fundamental for the development and progression of both localized and advanced metastatic prostate cancer ,11. AdvaThus, it is critical to investigate AR downstream-signaling or regulatory mechanisms by AR to understand how HRPCs/CRPCs develop among the patients. Therefore, identification of AR downstream signals and new molecular mechanisms for AR activation are important to improve the treatment of advanced prostate cancer . In the Although several studies have investigated alteration of miRNA expression profiles in prostate cancer tissues as potential diagnostic and prognostic tools, unique signatures are still missing. In 2006, genome-wide microarray analysis performed by Volinia et al. revealed a general upregulation of miRNAs in various cancer tissues . In contcaspase-7, E2F1 or p21 . Ou. Ou91]. gression . Meanwhigression . Moreovetastasis ,95. Impotastasis ,97. It wtastasis ,99. In aWe examined the expression of miR-29a and miR-29b in prostate cancer tissues. Our analysis revealed higher expression of these miRNAs in prostate cancer tissues is associated with poor prognosis of prostate cancer patients. In contrast, analysis of the miRNA signature of prostate cancer compared with benign tissues showed that expression of miR-29 family members including miR-29b is significantly downregulated in cancer tissues compared to normal prostate tissues . AlthougThe high stability of miRNAs  and abnoSec23 (S. Cerevisiae) Homolog A (SEC23A). In addition, we have also reported that miR-200 families are androgen-responsive as mentioned [Lin et al. investigated biomarkers by analyzing circulating miRNA in 97 CRPC samples. High levels of miR-21, miR-200 family  and low levels of miR-17 family  or miR-222 were associated with PSA-response and shorter survival of patients. High levels of miR-200 family members are associated with poor chemotherapy outcome of patients in this study . This grentioned , suggestIn addition, there is an increasing interest in the application of exosomes as non-invasive biomarkers . ExosomeBCL2, Cycline D1 (CCND1) and WNT3A in prostate cancer. Based on this mechanism, miR-16-conjugated atelocollagen has been shown to inhibit bone-metastatic human xenograft growth in mouse bone site in vivo [Effective delivery of miRNAs to cancer cells is very challenging because of the lack of miRNA trapping system to cancer cells . Moreove in vivo . In addi(1)AR-regulated miRNAs such as miR-21, miR-32, miR-125b, miR-141, miR-148a promoted tumor growth by regulating downstream signals such as cell cycle, apoptosis and invasion. These miRNAs are upregulated in CRPC or metastatic cancer.(2)AR or CD44 directly to inhibit development of tumor and cancer stem cells. Loss of these miRNAs may be critical step for prostate cancer progression.Several miRNAs target (3)Changes of global epigenetic code by AR-regulated miRNA induction would be important pathway for inducing HRPC and promotes tumor growth.(4)The functions of miRNAs such as miR-141, miR-221/222 and miR-29a/b are not unique during prostate cancer progression.The main priority in clinical prostate cancer research is identifying novel biomarkers to reliably distinguish between low-risk patients and high-risk patients who need definitive treatment. Strong evidences have been presented about the significance of miRNAs in prostate cancer (summarized in We should therefore consider the cell/tumor type or stage and expression levels of their target genes to evaluate the roles of these miRNAs. Although recent studies showed that measurement of these transcripts in clinical samples could improve the prediction of prognosis, more understanding of molecular events that lead to metastatic prostate cancer would enable us to determine treatment modalities at the time of diagnosis.


Schizophrenia is a chronic debilitating disorder which affects about 21 million people worldwide (WHO 2017). Elevated pre-synaptic striatal dopamine synthesis capacity is a robust neurochemical alteration seen in patients with schizophrenia compared to controls, with a large effect size Cohen’s d=0.79 . Ketamine, a non-competitive N-methyl-D-aspartate receptor (NMDAR) antagonist induces psychotomimetic effects in healthy human  and exacerbates psychotic symptoms in patients with schizophrenia . For these reasons, it has been used to model the neurochemical alterations seen in schizophrenia such as dopaminergic overactivity . However, the effect of sub-chronic ketamine on dopamine synthesis capacity in vivo is not known. Here we investigated the effect of sub-chronic ketamine on striatal dopamine synthesis capacity in vivo using Positron Emission Tomography (PET) imaging and on locomotor activity in the mouse. Moreover, via a chemogenetics approach (Roth 2016) we explored the role of midbrain dopamine neuron activity in mediating ketamine-induced effects.All procedures were conducted under licence in accordance with the UK Animals (Scientific Procedures) Act of 1986. Mice received a sub-anaesthetic dose of ketamine or an equivalent volume of saline for five consecutive days. Locomotor activity was assessed in the open field test. Moreover, mice received a dynamic 3,4-dihydroxy-6-[(18)F]-fluoro-L-phenylalanine Positron Emission Tomography (PET) scan to assess striatal dopamine synthesis capacity in vivo. Data were analysed using an extended Patlak graphical analysis approach . Further midbrain dopamine neurons were transduced with an adeno-associated virus vector expressing Gi-coupled (hM4Di) inhibitory receptors under the control of the dopamine transporter (DAT) promoter in DATCre positive mice. Standard immunohistochemistry was used to label dopamine neurons and mCherry expression in dopamine neurons was confirmed using confocal microscopy. Two weeks following the stereotaxic injection of the viral construct, mice received clozapine N-oxide (CNO) to study the effects of inhibiting dopamine neuron firing on locomotor activity and striatal dopamine synthesis capacity in the sub-chronic ketamine model. Data were analysed by two-tailed independent samples t-tests, one-way ANOVA and repeated measures two-way ANOVA followed by Bonferroni post hoc tests where appropriate. p<0.05 was considered statistically significant.Sub-chronic ketamine treatment significantly increased striatal dopamine synthesis capacity  and induced locomotor sensitization (p<0.01). hM4Di-mCherry viral construct was successfully transduced in midbrain dopamine neurons with over 98% specificity. Chemogenetic inhibition of midbrain dopamine neurons prevented the ketamine-induced elevation in striatal dopamine synthesis capacity  and locomotor sensitization (p<0.05).Our data show that sub-chronic ketamine results in the elevation in striatal dopamine synthesis capacity and locomotor sensitization and that these effects require midbrain dopamine neuron activation. Furthermore, our data are in support of the hypothesis that NMDA receptor hypofunction on GABAergic interneurons leads to disinhibition of glutamatergic projections and subsequently increase in dopamine neuron activity and dopamine synthesis capacity in projection targets such as the striatum.


Hepatocellular carcinoma (HCC) is the most common and aggressive type of malignant liver tumor. HCC progression depends significantly on its vascularization and formation of new blood vessels. Vascular endothelial growth factor A (VEGFA) is a crucial regulator of tumor vascularization and components of VEGF-induced cell signaling pathways are important targets of therapeutical drugs that demonstrated the highest efficiency in case of advanced HCC (sorafenib and regorafenib). VEGFA is expressed as a set of isoforms with different functional properties, thus VEGFA isoform expression pattern may affect tumor sensitivity to anti-angiogenic drugs. However, information about VEGFA isoforms expression in HCC is still incomplete and contradictory. The present study aims to quantitatively investigate VEGFA isoform expression aberrations in HCC tissue.A total of 50 pairs of HCC and non-tumor tissue samples were used to evaluate the VEGFA isoform spectrum using RT-PCR and quantitatively estimate changes in isoform expression using RT-qPCR. Correlations between these changes and tumor clinicopathological characteristics were analyzed.p < 0.001), while no uniform changes were detected for potent angiogenesis stimulators VEGFA-165 and VEGFA-121. Isoform balance in HCC shifts from VEGFA-189 towards VEGFA-165 or VEGFA-121 in the majority of cases (p < 0.001). Changes in fractions, but not expression levels, of VEGFA-189 (decrease) and VEGFA-121 (increase) correlated with advanced Tumor-Node-Metastasis (TNM) and Barcelona Clinic Liver Cancer (BCLC) tumor stages (p < 0.05), VEGFA-189 fraction reduction was also associated with poor tumor differentiation (p < 0.05).We identified VEGFA-189, VEGFA-165, and VEGFA-121 as predominant isoforms in liver tissue. Anti-angiogenic VEGFA-xxxb variants constituted no more than 5% of all mature VEGFA transcripts detected and their expression was not changed significantly in HCC tissue. We demonstrated for the first time that the least active variant VEGFA-189 is frequently repressed in HCC (A distinct shift in VEGFA isoform balance towards more pro-angiogenic variants occurs in HCC tissue and may modulate overall impact of VEGFA signaling. We suppose that the ratio between VEGFA isoforms is an important parameter governing HCC angiogenesis that may affect HCC progression and be used for optimizing the strategy of HCC therapy by predicting the response to anti-angiogenic drugs. Hepatocellular carcinoma (HCC) is the most common and aggressive form of primary liver tumor and ranks second place in cancer-related mortality rates . Most HCAngiogenesis is one of the most critical processes involved in HCC pathogenesis . It is mHigh levels of VEGFA expression were reported in different types of malignant tumors including HCC and are associated with advanced stages of disease, poor survival of patients and high recurrence rate . A numbeThus, evaluation of VEGFA isoforms expression pattern in tumor is important for a rational choice of anti-angiogenic therapy, which is considered to be a promising approach for HCC treatment. However, no quantitative analysis exploring tumor-specific alterations of VEGFA isoforms expression in HCC has been reported yet. The present study aims to compare the full spectrum of VEGFA isoforms expressed in paired HCC and non-tumorous (NT) liver samples and to quantitatively evaluate changes in the expression of major isoforms and their association with tumor clinicopathological characteristics.A total of 50 HCC tissue samples and 50 corresponding NT tissue samples were obtained from patients diagnosed with HCC after tumor resection, fresh-frozen in liquid nitrogen and stored at −70 °C. All patients were hepatitis-negative; additional data on clinicopathological parameters are presented in Reverse transcription was performed using total RNA isolated from 30 mg of tissue with PureLink RNA Mini Kit  using random hexanucleotides and RevertAid Reverse Transcriptase .Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of VEGFA isoforms expression spectrum was performed with VEGFA-iso primers flanking variable mRNA region . Primer Quantitative RT-qPCR analysis of VEGFA isoforms expression was performed using primers specifically detecting different VEGFA isoforms expressed in liver or isoform groups: VEGFA-total , VEGFA-xxxb, VEGFA-189, VEGFA-165, VEGFA-121  and S2. p < 0.05. When analyzing individual cases, twofold or stronger changes in gene expression were considered significant. Statistical analysis and graph plotting were performed using OriginPro 2016 software .Differences between observation groups were evaluated using paired sample sign test. Correlations were evaluated using Spearman’s rank test, for that numerical clinical parameters  were used as is, all categorical parameters were assigned rank values  indicating that VEGFA-189, VEGFA-165, and VEGFA-121 are indeed the main components of total VEGFA transcripts pool in examined specimens.VEGFA-189 is significantly downregulated in HCC sample set in the comparison to the NT sample set, while no significant changes were observed in VEGFA-165 and VEGFA-121 expression –2E. AnalSince different VEGFA-xxxa isoforms exhibit different angiogenesis-stimulating properties, a shift in their balance can considerably modulate VEGFA signalization. Thus, the cumulative impact of several VEGFA isoforms depends not only on their absolute expression levels, but also on proportions between particular variants. We therefore decided to estimate the ratios between VEGFA-189, VEGFA-165, and VEGFA-121. Since the amount of other VEGFA variants in examined samples is negligible in comparison with the amount of three predominant isoforms, we used simplified calculation model considering the amount of all mature VEGFA transcripts as a sum of VEGFA-189, VEGFA-165, and VEGFA-121 copy numbers in each individual sample and calculated fractions of each of the predominant isoforms See . A signiCorrelation analysis revealed that changes in VEGFA isoform fractions were stronger associated with clinically significant tumor characteristics than alterations in expression levels . IncreasThere is growing evidence that aberrations in alternative splicing play significant role in carcinogenesis. Splice isoforms may possess different functional properties defining their pro- or anti-oncogenic activity .Spectrum and ratios of expressed VEGFA isoforms are tissue-specific and provide the establishment of vascular network matching specific tissue functions . The difWhile VEGFA is known to be frequently overexpressed in various tumors , our resAnti-angiogenic VEGFA-xxxb variants, which are predominant in several normal tissues , comprisIn agreement with previously published data, VEGFA-189, VEGFA-165, and VEGFA-121 isoforms were the major variants expressed in non-cancerous liver . All thrAs stated above, the activity of VEGFA-induced signaling can be affected not only by changes in individual isoforms expression, but also by changes in their balance. The shift of the isoform ratio from poorly diffusible VEGFA-189 variants towards more angiogenic VEGFA-165 and VEGFA-121 ones observed in HCC tissue is much more prominent than alterations of expression levels of corresponding isoforms. There are two possible causes of VEGFA-189 fraction reduction: direct VEGFA-189 downregulation or increase in VEGFA-165 and VEGFA-121 expression levels. According to our results, these two alterations rarely occur simultaneously , thus imThe importance of VEGFA isoforms fractions evaluation is clearly illustrated by the fact that changes in VEGFA isoform fractions, but not in their expression levels, are associated with such essential HCC clinicopathological features as TNM and BCLC stages, the latter currently being considered the most effective system for HCC prognosis and treatment optimization . MoreoveIncrease in VEGFA-165 and VEGFA-121 expression levels is associated with ascites and feeble tumor capsule indicating their progression-stimulating role consistent with previously reported data . High leData on VEGFA-189 functions in different tumors are rather controversial. This isoform was reported to be overexpressed in colon, ovary and lung tumors, while its repression was described in non-small cell lung carcinoma . On the Current studies of the role of VEGFA splice variants in cancer development are mainly focused on balance of pro- and anti-angiogenic VEGFA isoform groups. At this time several regulators of VEGFA mRNA splicing have been identified  that interact with exon 8, thus controlling generation of VEGFA-xxxa or VEGFA-xxxb variants , 2010. HSince there is no reliable experimental approach to induce VEGFA splicing changes, most of published reports on functions of VEGFA-xxxa isoforms evaluate effects caused by overexpression of certain VEGFA variants . While iUsing a quantitative approach, we have detected HCC-specific shift of VEGFA isoforms ratios that consisted in decrease in VEGFA-189 and increase in VEGFA-165 and VEGFA-121 fractions. These changes were associated with multiple essential clinicopathological tumor characteristics. The clinical significance of presented data consists in their potent impact on optimization of HCC treatment since the VEGFA isoforms ratio may be a promising factor for prediction of anti-angiogenic therapy efficiency. Further studies of VEGFA isoforms expression, VEGFA mRNA splicing regulation and VEGFA-189 functional properties in HCC are necessary in order to evaluate its possible association with other tumor progression factors, survival and recurrence.10.7717/peerj.4915/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.4915/supp-2Supplemental Information 2“Unique sequence regions” represent fragments of PCR products identified by direct sequencing  that werClick here for additional data file.10.7717/peerj.4915/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.4915/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.4915/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.4915/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.4915/supp-7Supplemental Information 7Spreadsheets of the file contain raw data on fractions of VEGFA isoforms and TBP-normalized data on expression levels of VEGFA isoforms in all examined tissue samples. These data were used for plot generation and statistical analysis. More detailed information on each of spreadsheets is present in “Legend” sheet of the file.Click here for additional data file.10.7717/peerj.4915/supp-8Supplemental Information 8Lengths of DNA markers are indicated on the left, VEGFA isoforms are indicated on the right. The unmarked band under VEGFA-165 PCR product corresponds to heteroduplex of VEGFA-165 and VEGFA-121 PCR products but not to any certain VEGFA isoform, according to sequencing data .Click here for additional data file.10.7717/peerj.4915/supp-9Supplemental Information 9Data for all samples set (n=100) are presented as box-and-whisker plot.Click here for additional data file.10.7717/peerj.4915/supp-10Supplemental Information 10Data are presented as NT/HCC ratios in logarithmic scale.Click here for additional data file.