FDG-PET imaging studies are often performed on groups of rats, so it is desirable to establish an objective voxel-based statistical methodology for group data analysis.PET (positron emission tomography) imaging researches of functional metabolism using fluorodeoxyglucose  toolbox (plug-ins) named spmratIHEP for voxel-wise analysis of FDG-PET images of rat brain, in which an FDG-PET template and an intracranial mask image of rat brain in Paxinos & Watson space were constructed, and the default settings were modified according to features of rat brain. Compared to previous studies, our constructed rat brain template comprises not only the cerebrum and cerebellum, but also the whole olfactory bulb which made the later cognitive studies much more exhaustive. And with an intracranial mask image in the template space, the brain tissues of individuals could be extracted automatically. Moreover, an atlas space is used for anatomically labeling the functional findings in the Paxinos & Watson space. In order to standardize the template image with the atlas accurately, a synthetic FDG-PET image with six main anatomy structures is constructed from the atlas, which performs as a target image in the co-registration.The spatial normalization procedure is evaluated, by which the individual rat brain images could be standardized into the Paxinos & Watson space successfully and the intracranial tissues could also be extracted accurately. The practical usability of this toolbox is evaluated using FDG-PET functional images from rats with left side middle cerebral artery occlusion (MCAO) in comparison to normal control rats. And the two-sample t-test statistical result is almost related to the left side MCA.We established a toolbox of SPM8 named spmratIHEP for voxel-wise analysis of FDG-PET images of rat brain. Groups of human brain images can be statistical analyzed in SPM objectively and automatically, which is also desirable in rodent studies.PET (positron emission tomography) imaging techniques of functional metabolism using fluorodeoxyglucose  and FDG-PET images of small animals, automated image co-registration of FDG-PET data with MRI templates is difficult. Therefore, the established MRI template The extracranial tissues were unwanted in imaging data analysis. Recently, the intracranial tissues of each rat are usually traced out manually Furthermore, the assignment of anatomical location to functional effects is critical in the interpretation physiological significance of statistical result of voxel-wise analysis. For anatomical localization, an atlas space is prerequisite, such as the Tailarach space for human brain studies To align the template into the atlas space, Schweinhardt et al. shanbc@ihep.ac.cn.With the introduction above, the current study is designed to establish a voxel-wise analysis method of FDG-PET images of rat brain. In detail, an FDG-PET rat brain template, comprised cerebrum, cerebellum, olfactory bulb and extracranial tissues, will be constructed which is used for spatial normalization of individual rat brain images. An intracranial mask image in the FDG-PET template space will be constructed for extracranial tissues automatic removing. The FDG-PET template will be standardized into the Paxinos & Watson space for the localization of functional investigation. Finally, the constructed template sets are compiled as a SPM8 toolbox (plug-ins) named spmratIHEP. Moreover, to evaluate the usefulness of the toolbox implemented in SPM8, we analyze the data from rats with left side middle cerebral artery occlusion (MCAO) and those without. The spmratIHEP toolbox is available by contacting the corresponding author at 18F-FDG injection, but had access to drinking water at all time Twenty-four Sprague-Dawley (SD) rats of either sex, 10∼13 weeks old, weight 350 g±20 g, were used for template construction. A further ten similar rats were used for evaluation of spatial normalization and removal of extracranial tissues. Voxel-wise analysis of brain images was done on male SD rats, 9∼11 weeks old; weight 300 g±20 g; 9 underwent left side middle cerebral artery occlusion (MCAO) and 8 were healthy controls. Intraluminal occlusion of the middle cerebral artery (MCA) was accomplished using a modification of the Longa technique 18F-FDG was prepared at PET center of China PLA General Hospital. For each rat, 18F-FDG (18.5 MBq/100 g of body weight) was administered via tailvein injection without anesthesia. Then the rats were kept in their cages and placed in a room with minimal ambient noise for the 18F-FDG uptake. The uptake period was 40 min for maximization of 18F-FDG uptake in the brain For the twenty-four rats used in template construction, FDG-PET imaging was performed at the PET center of China PLA General Hospital in MicroPET/CT imaging system , of which the radial spatial resolution is 1.0 mm full-width at half-maximum (FWHM) at the centre of the field of view (FOV). During FDG-PET scan, all the rats were anesthetized with isoflurane (the same as described above) and placed in the scanner with a plastic stereotactic head holder in prone position on the scanner bed. And the rat brain was centered in the FOV to perform a static acquisition of 10 minutes. Images were subsequently reconstructed using 3D ordered set expectation maximization (3D-OSEM) algorithm. Corrections for dead time, decay, attenuation, random coincidences and scattering were applied. Images were reconstructed on a 175×175×61 matrix, where the voxel size equals 0.39×0.39×0.77 mm. All scans were saved as Analyze format.For the ten rats used for evaluation of spatial normalization and removal of extracranial tissues by the spmratIHEP toolbox, FDG-PET images were acquired on a micro PET system , of which the radial spatial resolution is 1.67 mm FWHM at the centre of the FOV. During FDG-PET scan, all the rats were anesthetized with isoflurane (the same as described above) and placed in the scanner with a plastic stereotactic head holder in prone position on the scanner bed. And the rat brain was centered in the FOV to perform a static acquisition of 30 minutes. Images were subsequently reconstructed using Filtered Back projection (FBP) algorithm. Images were reconstructed on a 128×128×63 matrix, where the voxel size equals 0.5×0.5×1 mm. All scans were saved as Analyze format.The nine rats with MCAO and eight ones without were used for evaluation of this toolbox spmratIHEP for its capacity for functional localization. Twenty-four hours after operation, FDG-PET images were acquired on Siemens Inveon PET , of which the radial spatial resolution is 1.4 mm FWHM at the centre of the FOV. During FDG-PET scan, all the rats were anesthetized with isoflurane (the same as described above) and placed in the scanner with a plastic stereotactic head holder in prone position on the scanner bed. And the rat brain was centered in the FOV to perform a static acquisition of 20 minutes. Images were subsequently reconstructed using FBP algorithm. Corrections for dead time, decay, attenuation, random coincidences and scattering were applied. Images were reconstructed on a 128×128×159 matrix, where the voxel size equals 1.4×1.4×0.79 mm. All scans were saved as Analyze format.All experiments were performed with the approval of the Animal Care and Use Committee of the Chinese Academy of Sciences and conformed to named international guidelines on the ethical use of animals.The FDG-PET rat brain template image with the extracranial tissues was created using SPM8. All of the 24 images included in this study were inspected and were found equally of high quality in terms of the image contrast, noise level, and resolution. Firstly, the voxel size of all the brain images of 24 rats were adjusted to [1 1 1.8], which was scaled up in the Analyze header by the factor of 4 to better approximate human dimensions i is the running voxel position index  in the image matrix, and n is the total number of voxels. Then the original 24 images were recursively registered to the new average target image. A newer average target image was created from the newly registered images. And the mean squared residual difference was subsequently computed between the current target image and the previous one. This procedure, including registration, averaging and computing the mean squared residual difference, was repeated until the latest mean residual squared difference was stabilized (less than 5% in this current study) The 24 spatially normalized images were averaged to create a new template. The mean squared residual difference between the old and new templates was computed. The mean squared residual difference is defined as in Eq. (1).The canonical brain was created by extracting intracranial voxels from the average template image constructed above. We adopted the global histogram threshold method to exclude voxels outside the rat brain. The threshold was determined by Otsu’s criterion Moreover, this extracted canonical brain was processed by binarization transformation to create an intracranial mask image in the standard template space. The origin point of the canonical brain and intracranial mask was the same with the template. Thus, after normalizing to the FDG-PET template, the extracranial tissues of individuals could be automatically removed by this intracranial mask image.bregma +7.56 mm) to the posterior part of cerebellum (zbregma −15.72 mm). In order to co-register the canonical FDG-PET brain with the MRI structural template and atlas, we created a low-resolution synthetic PET image from the atlas 3 FWHM to smooth the juncture of the neighboring segmented parts.In this study, the paired rat brain structural MRI template and digital atlas in Paxinos & Watson space we constructed before rd ventricle (D3V).The synthetic FDG-PET image was chosen as the target image. The SPM intensity-based affine transformation algorithm and subsequent non-linear warping algorithm was employed to co-register the canonical brain with this target atlas image In order to analyze rat brain images with less manual manipulation, we constructed a SPM8 plug-ins toolbox named spmratIHEP using MATLAB. Because of the differences between human brain and rat brain, the processing methods and parameter settings in SPM8 must be modified according to the feature of rat brain FDG-PET imaging, which were described in detail as below.Firstly, spatial normalization is prerequisite in voxel-wise analysis to eliminate individual differences. In order to use the available affine transformation and subsequent non-linear warping algorithm in SPM8, the voxel size of individual brain images was scaled up in the Analyze header by a factor to better approximate human dimensions, which will not affect any interpretation of the statistical results of rat brain.Then, the constructed FDG-PET template and intracranial mask of rat brain took replacement of the human brain. And the default parameter settings of spatial normalization were adjust according to the feature of rat brain, that the bounding box was adjust to , the voxel size was adjust to [1 1 1.8] after zooming and the affine regularization was adjust to ‘average sized template’. Moreover, the image matrix of intracranial rat brain images after spatial normalization was sheared to [120 80 98] automatically to cut off the background.3 FWHM, which was approximately two times of the spatial resolution of rat brain after zooming Smoothing was used to improve the signal-to-noise ratio in SPM, in which the Gaussian kernel was adjust to 2×2×4 mmFinally, in order to display the statistical results of rat brain in Paxinos & Watson space, the definition of coordinates of rat brain took replacement of the human brain. The accurate location of the maximum FDG changes was modified according to the Paxinos & Watson space, in which the x-axis is negative to the left from the midline and positive to the right, the y-axis is positive to the ventral direction relative to the dorsal, and the z-axis is positive to the olfactory bulb direction relative to the bregma and negative to the cerebellum direction. Moreover, a new projection figure of rat brain was created to show the projection of overall blob regions, and a MRI T2-weighted (MRI T2WI) structural image of a single rat brain in Paxinos & Watson space was prepared to show the three-dimensional view of a blob region.3 FWHM.The analysis of MCAO rat model images was performed using our spmratIHEP toolbox to evaluate its practical use. Firstly, the body tissues and background of individual images were manually removed using MRIcro Then, the preprocessed images were analyzed based on the framework of the general linear model (GLM). Two-sample t-test was performed to identify the difference of FDG signals between the rats with MCAO and the healthy controls, in which proportional scaling and intensity normalization was applied to account for global confounds. Finally, the brain regions with significant FDG changes in rats with MCAO were yielded based on a voxel-level height threshold of p<0.05 (FWE corrected) and a cluster-extent threshold of 50 voxels.3 after zooming. This template set was consisted of the standard FDG-PET template with extracranial tissues . Then, four volumetric and spatial correspondence measures, Jaccard similarity (JS) To illustrate the successful use of the intracranial mask image in removing non-brain tissues automatically, both datasets obtained for template construction and spatial normalization evaluations were used. The individual images from both datasets were spatially normalized in spmratIHEP. The extraction of three randomly chosen rats result was shown in The Jaccard similarity was defined as in Eq.(2), of which the optimal value is 100%.Where The relative error on volume was defined as in Eq.(3), of which the optimal value is 0%.Where The proportions of false-positive was defined as in Eq.(4), of which the optimal value is 0%.Where The proportions of false-negative was defined as in Eq.(5), of which the optimal value is 0%.Where The calculated results are given in The strategy we proposed to co-register the FDG-PET rat brain template with the atlas via synthetic FDG-PET image worked well. The pseudo FDG-PET with six major structures is shown in For qualitatively evaluation, the co-registered FDG-PET canonical brain is overlaid on the MRI T2WI structural canonical brain which has already standardized into Paxinos & Watson space in our prior study E’ stands for the voxel numbers in each cluster, the ‘PFWE_coor’ stands for the maximum confidence level in each cluster, the ‘Max_T’ stands for the maximum t-value in each cluster, the ‘Max_Z’ stands for the maximum Z-value in each cluster, the ‘Peak coordinates (mm)’ stands for the coordinates of the maximum point in Paxinos space. These regions are exclusively related to the left side MCA.The analysis of MCAO rat model images was performed using our spmratIHEP toolbox to evaluate its practical use. The statistical result that brain regions with significant FDG declined in rats with MCAO was shown in a projection figure  and fuseIn the current study, we established a SPM8 plug-ins toolbox named spmratIHEP for voxel-wise analysis of FDG-PET images of rat brain, in which an FDG-PET template and an intracranial mask image of rat brain in Paxinos & Watson space For voxel-based statistical analysis of functional images, all the individual images should be primarily normalized into one common space, such as the MNI  space in human brain studies. And the standard brain template performs as a general criterion in spatial normalization. In our study, to spatially normalizing individual rat brain images, an FDG-PET rat brain template was iteratively created from 24 healthy adult rats. Because there are minor differences in anatomical structures between healthy ones How to locate the anatomical regions of functional effects in statistical results is important in voxel-wise analysis. In physiological significance location, the brain atlas also called stereotaxic coordinates corresponding to the template space provides detailed anatomical information for functional results, such as the Tailarach coordinates for human brain studies In addition, to evaluate the performance of spmratIHEP in voxel-wise analysis of FDG-PET images, nine rats with left side MCAO and eight healthy controls, acquired on Siemens Inveon PET which differs from the one imaging for template construction, were enrolled in this study. The data preprocessing and voxel-wise analyzing of these two groups were all performed automatically in spmratIHEP based on SPM8. As illustrated in the Result part, the statistical result was almost related to the left side MCA. Therefore, our constructed toolbox performed well in rat brain imaging data analyzing in SPM8.In conclusion, demonstrating its adequacy and practical usage in SPM environment, we reported the constructed FDG-PET rat brain template in the stereotaxic coordinate space of the Paxinos and Watson rat brain atlas. We believe it will be helpful to streamline the neuroimaging data analyses for rat brain images from the pre-processing stage to the reports of the statistical inference results.


The presence of bone metastasis is a relevant prognostic factor for cancer patients, andthe skeleton is a common site of distant metastasis in cases of advanced-stage neoplasias.Recent developments in oncological treatments have led to a significant increase inpatients’ survival. On the other hand, much of the morbidity and disability in suchpatients is caused by metastatic bone disease. Thus an appropriate assessment of theskeleton for early detection of bone metastasis is essential for correct clinical decisionsmaking in order to provide patients with appropriate treatment and better quality oflife.99mTc-labeled radiopharmaceuticals, such as99mTc-MDP, has been extensively utilized for the diagnosis of bonemetastasis, and plays a relevant role in the initial assessment and follow-up of cancerpatients. In spite of its relatively high sensitivity, the accuracy of BS is compromised byits limited spatial resolution. Thespecificity is frequently low as the skeleton may be affected by different diseases with nospecific scintigraphic findings. Forthis reason, about one third of BSs present inconclusive results for bone metastasis, whichimplies the necessity of additional procedures for a definite diagnosis or for anatomicalcorrelation, namely radiography, computed tomography, magnetic resonance imaging andbiopsy,4. The routine use of tomographic images such as SPECT/CT (singlephoton emission computed tomography/computed tomography) is also helpfull, but itsignificantly increases imaging acquisition time, particularly in cases where severalsegments of the skeleton must be scanned.Bone scintigraphy (BS) with 18F-NaF) has been utilized in theassessment of bone metastasis in a variety of malignancies-6. The fast andintense 18F-fluoride uptake by active osteoblastic lesions and by theosteoblastic component of osteolytic lesions occurs due 18F-fluoride ionsexchange with the hydroxyl groups in hydroxyapatite crystals. The method accuracy is quitehigh and several studies demonstrate that 18F-fluoride PET/CT is more sensitiveand specific than BS to identify bone metastases due its higher spatial resolution and tothe more favorable biokinetics of the radiopharmaceutical-6.Positron emission tomography/computed tomography (PET/CT) with the radiopharmaceuticalsodium fluoride labeled with fluorine18 . Exceptfor Ipen, all the other ten centers involved in the production of18F-fluorodeoxyglucose (18F-FDG) do not commercialize18F-fluoride, despite the simplicity of its production. Certainly, the mainreason for that is the low demand for 18F-fluoride PET/CT scan since it is notcovered by both public and private health care systems. In fact, the cost-effectiveness of18Ffluoride PET/CT is still to be established, particularly in Brazil. Thecost of the radiopharmaceutical 18F-fluoride is higher than that ofMDP-99mTc utilized in BS and, in addition, the operational cost of a PET/CTprocedure is also higher than that of a scintillation camera. In this context, the timespent to perform a BS or a 18F-NaF PET/CT becomes one of the main factors thatcould balance the overall costs involved in the performance of each of the two procedures.Such an issue is the core of an extensive and definitive study developed by Ordones et al.and published in the previous issue of Radiologia Brasileira.In Brazil the availability of 18F-fluoride PET/CT scans as a means to reduce imaging acquisitiontime by 25% in the investigation of bone metastases. As much as one thousand whole-body18F-fluoride PET/CT scans were reviewed by the authors and only three casesof bone metastases exclusively in the lower limbs were observed; and one of such lesionswas not a true bone metastasis, rather, it was a soft tissue metastasis infiltratingadjacent bone tissue. Thus, the prevalence of this type of lesion corresponded to only0.2%. Also, it is important to highlight that the two mentioned true exclusive lower limbmetastasis found by the authors would have been detected at images acquired up to the rootof the thighs, which has already been traditionally done and accepted for many yearsworldwide for most indications of 18F-FDG PET/CT scans.The authors sought to establish the possibility of excluding lower limbs images fromwhole-body 18F-fluoride PET/CT scan can be initiated 30-60 minutesafter radiopharmaceutical administration. An acquisition up to the proximal femurs asproposed by Ordones et al. does nottake more than 12 minutes to be completed. Therefore, it might take less than one hourbetween 18F-fluoride injection and the patient discharge after imageacquisition. It is possible to perform PET/CT scans in up to four patients per hour, whichcorresponds to at least twice the number of BSs performed during an equal period of time,particularly in cases where SPECT images are acquired. The acquisition of additionalimages, frequently needed in BSs, is almost never necessary in a 18F-fluoridePET/CT scan, and the procedure is completed with all the possible correlations with CTalready performed – at least for anatomical correlation -, which considerably reduces thenecessity of other imaging procedures. As noted by the authors, time savings also provideradiopharmaceutical savings, since 18F half-life is < 2 hours .Generally, a BS is obtained three hours after radiopharmaceutical administration, andwhole-body and additional static images acquisition takes about 30 minutes to be completed.As a SPECT or SPECT/CT acquisition is added, the patient remains in the examination roomfor at least additional 15 minutes. As necessary, further correlation with radiography, CTor magnetic resonance imaging is also performed, thus generating additional time and costs.On the other hand, a 18F-fluoride PET/CT is considered to be the most comprehensiveimaging modality to evaluate metastatic bone disease. The inclusion of such imaging method in the oncological routinedepends on the establishment of practical and effective protocols whose costs areacceptable by the health care system. Therefore, it is clear the relevance of the studydeveloped by Ordones et al. as a support for the incorporation of 18F-fluoridePET/CT imaging into the clinical practice.Currently,


Interleukin-2 (IL-2) therapy has been used with success in curing metastatic renal cell carcinoma and melanoma in a small minority of patients. However, the benefits can be accompanied by severe toxicity. This review of the literature discusses varying doses of IL-2 and their associated response rates and the toxicities associated with treatment. The review also explores the maximally beneficial dose with the most tolerable side effects. Although the higher-dose regimens with a more frequent dosing schedule produce higher-grade toxicity, they were found to deliver the most durable and complete responses. It is recommended to use a higher-dose regimen  and provide supportive care for toxicity, so patients can have maximal benefit from therapy. The administration of high-dose intravenous interleukin-2 (IL-2) for metastatic renal cell carcinoma and metastatic melanoma was first approved by the US Food and Drug Administration (FDA) in 1992 and 1998, respectively ., 2005. The use of high-dose IL-2 in renal cell carcinoma and melanoma are two rare instances in oncology where an effective treatment has been identified to potentially cure a widely metastatic solid tumor . The useThere are three identified classes of IL-2 receptor complexes, with a high, intermediate, or low affinity for binding with IL-2 . It can Varying levels of systemic toxicity are expected in patients who receive IL-2. There is evidence that increased doses of IL-2 lead to increased toxicity, which is not tolerable to all patients . Side efThe current FDA-approved dose of high dose IL-2 is 600,000 IU/kg per dose administered intravenously every 8 hours for a maximum of 14 doses on days 1 to 5 (cycle 1) and days 15 to 19 (cycle 2), with a maximum of 28 doses for 1 course . The dosing is discontinued when the patient has reached a dose-limiting toxicity that would compromise the patient’s safety.Several dosing schemes, which include intravenous high dose , low-dose subcutaneous injections, and IL-2 in combination with other treatments, have been used in practice for maximal therapeutic benefit. The variety of regimens makes it difficult to determine the most effective and least toxic option for the treatment of metastatic renal cell carcinoma and melanoma. There are currently 60 institutions in North America that administer high-dose IL-2 therapy for metastatic melanoma and renal cell carcinoma ., 2014.Therefore, the purpose of this comprehensive review is to examine the use of IL-2 at different dosing regimens in metastatic renal cell carcinoma and melanoma to determine whether there is a target dose that produces optimal patient outcome with minimal toxicity.We identified relevant articles in the Scopus and PubMed databases utilizing the search terms "interleukin-2," "IL-2," "toxicity," "response," "melanoma," and "renal cell carcinoma." There were 363 results that were populated. Limitations imposed within the search were for articles published between January 2002 and October 2014, English language, peer-reviewed journal articles, and human populations. Articles were excluded if upon review they were not clinically relevant to adult patients receiving IL-2 for renal cell carcinoma or melanoma. Case studies and review articles were excluded, as were articles that focused specifically on one area of metastasis. Studies that focused on one population (geriatrics or pediatrics) were also excluded. This comprehensive review includes 10 articles that met these search criteria. The data extracted from the articles include the protocol for administering IL-2 with the dosing schema. The reported toxicities and response rates were also extrapolated. The scales and measures for reporting toxicities and response were evaluated for their validity.Patient ToxicitiesCommon toxicities of high-dose intravenous IL-2 are included in Capillary leak syndrome is a life-threatening toxicity resembling septic shock that may occur with intravenous high-dose IL-2. The increased IL-2 in the circulation and immune stimulation cause may massive cytokine release and inflammatory reaction. Capillaries become more permeable, leading to the loss of intravascular fluid into extravascular spaces  intravenous treatment were not trivial; more than half of the treatment courses were discontinued due to intolerable side effects. The two most common reasons for discontinuation of low-dose intravenous treatment were hypotension and patient refusal ., 2003.Adding IL-2 to chemotherapy  has proved to be more toxic than chemotherapy in two trials. Three studies reported patient death attributable to IL-2 therapy. Patient OutcomesThe primary outcome of response to treatment was defined by various endpoints such as disease-free progression, total survival, or factors correlated with response. In solid tumors, such as melanoma and renal cell carcinoma, a standard to measure response to treatment is computed tomography (CT) or magnetic resonance imaging (MRI). Tumor measurement by radiologists requires a standardized method and documentation to allow for reproducibility.Criteria for measuring response varied from study to study. The World Health Organization (WHO) tumor response criteria were widely used to standardize independent image review for oncology studies. Subsequently, Response Criteria in Solid Tumors (RECIST) was considered to better refine measurement criteria that could be compared between studies ., 2000. In the data reported by High-dose IL-2 is a regimen ranging from 600,000 IU/kg to 720,000 IU/kg every 8 hours, and a low-dose IL-2 regimen is anything below this dosing protocol. Low-dose therapy was shown to have some response in patients; however, it rarely led to a complete response. When chemotherapy alone was compared with chemotherapy and IL-2, no survival advantage for the BCT arm was observed ., 2008. In the comparison of two high-dose therapy regimens , In a review of 259 patients, In comparing dosages of IL-2, the goal is to determine a dose that will provide the most benefit with the least amount of toxicity. For a small group of patients for whom cure is possible from metastatic disease, IL-2 is promising; however, in attempting to reduce toxicity, some regimens may negate the possibility of response. Our review of the literature suggests that high-dose IL-2 with supportive care for acute toxicity in the period immediately after its administration is the optimal dosing regimen with the most durable and complete response rates.However, it is difficult to evaluate a durable complete cure rate when rates are below 10% and lesser benefit is not evidenced by a significant proportion of the remaining patients ., 2003. Virtually all toxicity seen with intravenous administration of high-dose IL-2 is reversible and dose-limited ., 2003. There is no evidence to support a correlation between the severity of side effects and a higher propensity to respond to treatment . Alwan eThe differences between studies may be due to differences in methodology, including the fact that There may be practical reasons why high-dose IL-2 is not favored at particular institutions. The administration of high-dose IL-2 requires a monitored setting and possible transfer to an intensive care unit, a potential disadvantage to hospital management, as it requires more resources than routine cancer treatment ., 2008. Although very low-dose regimens may circumvent some toxicity, there is appropriate concern that reducing the schedule may limit response rates or response durations . It is iAdditionally, there is great variation in the toxicities that present in IL-2 patients, as some experience very few side effects . There mAdvanced practitioners may screen patients for appropriate therapy in the outpatient setting and assess patients at the bedside for administration of IL-2. The recognition of expected adverse reactions and proper interventions to prevent extreme toxicities are vital pieces of therapy ., 2001. To determine the optimal dosing of IL-2, a randomized controlled trial is necessary to overcome bias and detect a difference in responders and nonresponders, as the percentage of patients who receive benefit from this treatment remains low. Additionally, little is known about prognostic factors that can be linked to IL-2 response. Further research is needed to understand how IL-2 produces a complete and durable response in a small group of patients with metastatic melanoma and renal cell carcinoma and fails to stop even disease progression in others. There are currently no biomarkers or prognostic factors that have been identified to determine patients who may receive maximal benefit from high-dose IL-2 therapy, an important consideration when subjecting a patient to a treatment with profound side effects ., 2014. The treatment for melanoma has included several new immunotherapies, each bringing with it a host of adverse effects. Adverse events related to these agents were reviewed by Ma and Armstrong (2014), and unique adverse effects related to ipilimumab (Yervoy), vemurafenib (Zelboraf), interferon, dacarbazine, and IL-2 were found. This is noteworthy for advanced providers to be aware of the adverse effects that may be associated with therapies used as alternatives to IL-2; patients may still find side effects or toxicities intolerable, limiting their ability to receive full dosing .In recent years, targeted therapy has become more prevalent in oncology, and agents have been developed to treat melanoma and renal cell carcinoma. As a result, discussions on the value of IL-2 in comparison with these more focused treatments have emerged. Tyrosine kinase inhibitors used in renal cell carcinoma have increased the median survival by 10 months but have failed to produce consistent long-term durable responses. The cytotoxic T-lymphocyte antigen 4 inhibitor ipilimumab has been used in metastatic melanoma and has shown an increase in survival when compared with vaccine trials; however, it has been associated with significant autoimmune toxicity . For melanoma patients with a BRAF V600E mutation, vemurafenib has shown a complete response rate of 6% in a phase II trial, with a median response rate of 6.7 months .Compared with other treatments, high-dose IL-2 has consistently delivered durable complete responses of 10% to 15% in melanoma patients and 15% to 25% of patients with renal cell carcinoma. With properly trained practitioners and nursing staff, IL-2 should still be considered a first-line treatment for patients with renal cell carcinoma. Additionally, it is not known whether front-line treatment with another modality will change the action of IL-2 in the body ., 2014. High-dose IL-2 is a chance for cure for patients with metastatic renal cell carcinoma or melanoma. The factors and profiles of patients that increase this chance for cure remain elusive. Advanced practitioners should be aware of the toxicities associated with treatment to manage patients through administration and enhance the opportunity for them to receive the maximal amount of therapy.AcknowledgmentThe authors thank Edward Carney, Jr., for his administrative assistance in the preparation of this manuscript.


Studies have revealed that prolonged, specialized training combined with higher cognitive conditioning induces enhanced brain alternation. In particular, dancers with long-term dance experience exhibit superior motor control and integration with their sensorimotor networks. However, little is known about the functional connectivity patterns of spontaneous intrinsic activities in the sensorimotor network of dancers. Our study examined the functional connectivity density (FCD) of dancers with a mean period of over 10 years of dance training in contrast with a matched non-dancer group without formal dance training using resting-state fMRI scans. FCD was mapped and analyzed, and the functional connectivity (FC) analyses were then performed based on the difference of FCD. Compared to the non-dancers, the dancers exhibited significantly increased FCD in the precentral gyri, postcentral gyri and bilateral putamen. Furthermore, the results of the FC analysis revealed enhanced connections between the middle cingulate cortex and the bilateral putamen and between the precentral and the postcentral gyri. All findings indicated an enhanced functional integration in the cortico-basal ganglia loops that govern motor control and integration in dancers. These findings might reflect improved sensorimotor function for the dancers consequent to long-term dance training. Dance is a complex rhythmic sensorimotor activity that integrates organized spatial pattern body movements, temporally synchronized with timekeepers and other dancers, and adheres to a trajectory map of the body in exocentric space1There are two core elements in dance: metrical movement and organized spatial patterns14467899Using neuroimaging, brain activity during dance performance has been observed in the superior temporal gyri (reflects the auditory processing of music), the basal ganglia , the thalamus (relating to the connection between somatosensory and motor parameters), and the sensorimotor areas 11013151712Resting-state functional connectivity (rs-FC), which is based on the association of intrinsic spontaneous low-frequency blood-oxygenation-level dependent (BOLD) signal fluctuations between spatially remote brain regions, is an excellent approach with which to study brain plasticity in humans212324Based on prior studies, this study aimed to show that dancers had better motor control and better-integrated sensorimotor system when compared with non-dancers. We thus hypothesized that the dancers might show increased functional connections in sensorimotor brain networks that are related to dance properties. As such, we designed a cross-sectional experiment (dancers and non-dancers) and used rs-FC to test our hypothesis. FCD analysis and rs-FC analysis were performed to assess functional differences in sensorimotor systems induced by dance training. The purpose of these analyses was to find evidence of enhancement and integration of the various parts of the human brain linked to the sensorimotor movement of the dancers, which might be associated with the long-term effects of engaging in dancing.Mage = 19.07 ± 1.82 yrs)-henceforth referred to as the “dancer group”-and 33 non-dancing individuals -the “non-dancer group.” The participants in the dancer group consisted of college students majoring in modern dance at the Southwest University for Nationalities and the University of Electronic Science and Technology of China (UESTC). All of the subjects were assessed by a group of senior dancers. Subjects who qualified according to the admission requirements for a professional modern dance student were defined as dancers. The dancers engaged in regular dance training for an average of 12 hours per week with training occurring year-round over an extended period of time ranging from 7-17 years. We excluded subjects whose training periods were too irregular as determined by a questionnaire survey. Details about the subjects in the dancer group are provided in p = 0.96, according to a chi-square test; for age, p = 0.33, according to two-sample t-tests; for education, p = 0.16, according to two-sample t-tests). All subjects were right-handed according to the Edinburgh InventoryTwo groups of test subjects were recruited for this study, including (1) 28 dancers , with an eight-channel phased array head coil. All subjects underwent a 510-second resting-state scan to yield 255 volumes (32 slices per volume). None of the subjects performed a dance piece prior to the fMRI imaging. During resting-state fMRI, all subjects were instructed to be eye-closed and to move as little as possible without falling asleep. Subsequently, high-resolution T1-weighted images were acquired using a 3-dimensional fast spoiled gradient-echo (T1-3D FSPGR) sequence  = 1 mm, 152 slices).Images were acquired on a 3T MRI scanner (GE DISCOVERY MR750) at the MRI Research Center of UESTC. During scanning, we used foam padding and ear plugs to reduce head motion and scanning noise, respectively. Resting-state functional MRI data were acquired using gradient-echo echoplanar imaging(EPI) equences  software. A series of preprocessing steps were performed: (1) discarding five volumes to establish the magnetization equilibrium; (2) slice-timing correction; (3) head-motion correction; (4) normalization of images with an EPI template in the Montreal Neurological Institute (MNI) atlas space 3; (5) temporal filtering (band-pass 0.01-0.08 Hz); and (6) regressing nuisance signals (six motion parameters). The general processing procedures of spatial smoothing  of 8 mm) and regressing nuisance signals  were not included for the FCD analysis but were included for the functional connectivity analysis. Any subject who had a maximum displacement in any of the cardinal directions larger than 1.5 mm or a maximum spin larger than 1 degree were excluded from subsequent analyses. In addition, we also assessed translation and rotation in both groups using the following formula: M = 250 in this study). ix, iy and iz are translations/rotations at the ith time point in the x, y, and z directions, respectively, Δxid = ix–i–1x, and similar for iy and iz.Data processing was carried out using SPM8  between this voxel and all other voxels in the brain. Therefore, the short-range FCD of a given voxel reflected local functional connectivity, and the long-range FCD reflected functional connectivity with distant regions. Finally, individual normalized short-and long-range FCD (k-score) maps were obtained through division by the mean value of each individual map and spatial smoothing using 8 mm FWHM, respectively. Voxel-wised two-sample t-tests calculated in SPM8 were used to evaluate group differences in short- and long-range FCD maps between the groups.According to the approach introduced by Tomasi and Volkaw2727z-transformation. In this manner, functional connectivity maps of these seeds were produced for each participant. Voxel-wise two-sample t-tests calculated in SPM8 were used to determine differences in functional connectivity of the seeds between the two groups. Additionally, based on the analysis of the FCD and FC, the region-wise functional connectivity analysis and classification method were used in the following analyses  as the seeds. The mean BOLD time series were extracted from the seeds. Subsequently, functional connectivity analysis was performed between the seed and all voxels in the brain. The resulting correlation coefficients were transformed to approximate a Gaussian distribution using Fisher’s analyses .z-score of the ROI in which altered functional connectivity (FC) with those seeds was observed. Each ROI’s average z-score was correlated with the dance-training variables.Pearson’s correlation analysis was used to investigate the relevance to dance learning of the altered functional brain properties of the dancer group. First, we correlated the dance-training variables  with the k-scores of the FCDs in regions of interest (ROI). For findings of functional connectivity of the seeds with different FCD regions, we extracted the average T = 1.31, P = 0.19 for translational motion, and T = 1.22, P = 0.23 for rotational motion).None of the subjects was excluded according to the head-motion criterion. There were no significant differences between the two groups with regard to head motion or rotation  and long-range FCDs in the bilateral putamen and right occipital cortex significantly increased. Through the use of seed-based functional connectivity analyses for five regions , we further investigated specific functional connectivity. Compared with the non-dancers, the dancers showed significantly increased functional connections between the putamen and the middle cingulate cortex (MCC), as well as increased sensorimotor region functional connectivity with the precentral gyri and the postcentral gyri , Fig. 2.In the dancer group, we found significant positive correlations between the average training time per week and mean FCD k-scores of ROIs . FunctiOur findings identified improved functional connectivity in cortico-basal loops in the brains of dancers who had gone through intensive dance training for years. First, we found significantly increased short-range FCDs in the primary sensorimotor cortex and long-range FCDs in the bilateral putamen in dancers compared with non-dancers. Second, the dancers demonstrated significantly increased putamen functional connectivity with the MCC and connectivity in the sensorimotor cortices. Finally, the behavioral data analysis revealed positive relationships between functional properties and the average training time per week. These findings provided evidence supporting the notion that long-term dance training may be related to enhanced functional integration in cortico-basal ganglia loops.Similar to the regimented motor training required of athletes, dance training includes massive motor execution. When dance movements are performed, internal processing of kinesthesia occurs, and increased use of the muscles is actuated by the sensorimotor cortex. We found increased short-range FCDs and enhanced functional connectivity in the sensorimotor cortices  and primary somatosensory cortex (S1), which take charge of the forces responsible for muscle contraction in initial and final positions in dance and in basic sensorimotor activities, serve as areas important in motor execution and prim1734363839et al. reported enhanced activations in this loop in dancers and presumed that these activations might help with the organization and integration of segments of action and interactions among entrainment, meter and spatial patterning in dance21Different from regimented motor training, dance training can lead to profound changes in the functional organization of the sensorimotor cortices because of the need for artistic fine motor skills434345Most dance is performed to musical rhythms. The entrainment of dance to musical rhythms involves not only coherence from a temporal perspective but also a synchronization to the motor pattern in metric music bits, which is a unique trait of dance491549501Some limitation of the study should be mentioned. On one hand, the threshold (Tc) of the correlation coefficient to define the functional connection was set at 0.6 in the FCD analysis based on prior knowledge, and this fixed value may lead to some false positive or negative findings. Flexible thresholds, such as a set of continuous thresholds, should be considered in the future. On the other hand, the differences in the motor-related networks identified in this study may simply reflect the altered coherence characteristic of a resting state and may not directly predict dancer behavior. This is also an issue for all resting-state studies and requires further investigation. Multimodal and longitudinal designs may be useful in future investigations. In the current study, a cross-sectional design based on rest fMRI was adopted, which showed brain differences between the two groups. This design faced difficulties in its aim to reflect the brain plasticity resulting from dance training. Further longitudinal research should be included in the future.In conclusion, our findings reflect the enhanced functional connections in sensorimotor-related brain networks that exist in dancers who engage in prolonged modern dance training. The dancers presented with enhanced functional integration of cortico-basal loops and three biomarkers for optimal group separation with non-dancers, supporting the notion that improved motor execution and integration exists in dancers. In addition, the sensorimotor FC might predict the intensity of dance training. Moreover, the findings involving the putamen, which were closely related to rhythmical movement, indicate enhanced function related with dance properties. The findings derived from these tests offered substantial proof that the cortical ganglia loops of dancers operate at notably higher levels than for non-dancers. Although the results of this study were not outside the normal range compared to the existing research, they did contribute to our understanding of the benefits of dancing on the human brain.Furthermore, dance is much more than a complex sensorimotor movement. It includes the experience and expression of emotions, the pleasure of esthetics and interpersonal communication. Our present research showed no evidence of esthetic or emotional intrinsic differences for dancers. These unconfirmed traits of dance may be a rich and promising field in future study.How to cite this article: Li, G. et al. Identifying enhanced cortico-basal ganglia loops associated with prolonged dance training. Sci. Rep.5, 10271; doi: 10.1038/srep10271 (2015).


Introduction. In a previously published article the authors reported the long-term follow-up results in 138 consecutive patients with gallstones and common bile duct (CBD) stones who underwent laparoscopic transverse choledochotomy (TC) with T-tube biliary drainage and laparoscopic cholecystectomy (LC). Aim of this study is to evaluate the results at up to 23 years of follow-up in the same series. Methods. One hundred twenty-one patients are the object of the present study. Patients were evaluated by clinical visit, blood assay, and abdominal ultrasound. Symptomatic patients underwent cholangio-MRI, followed by endoscopic retrograde cholangiopancreatography (ERCP) as required. Results. Out of 121 patients, 61 elderly patients died from unrelated causes. Fourteen patients were lost to follow-up. In the 46 remaining patients, ductal stone recurrence occurred in one case  successfully managed by ERCP with endoscopic sphincterotomy. At a mean follow-up of 17.1 years no other patients showed signs of bile stasis and no patient showed any imaging evidence of CBD stricture at the site of choledochotomy. Conclusions. Laparoscopic transverse choledochotomy with routine T-tube biliary drainage during LC has proven to be safe and effective at up to 23 years of follow-up, with no evidence of CBD stricture when the procedure is performed with a correct technique. Laparoscopic cholecystectomy (LC) is the gold standard treatment for symptomatic cholecystolithiasis. The most appropriate management of concurrent common bile ductal stones (CBDS) in the elective setting, however, remains controversial. CBDS are present in approximately 10–15% of patients undergoing cholecystectomy for symptomatic gallstones  and its The improved laparoscopic skill and the development of dedicated laparoscopic instrumentation have offered the opportunity to treat gallstones and CBDS laparoscopically during the same session. This option, which has been introduced since more than 20 years into routine surgical practice and has become routine in a few centers, has proven to be a safe and effective alternative to the sequential endolaparoscopic approach. The EAES clinical trial comparing LC and common bile duct exploration (LCBDE) versus ERCP with endoscopic sphincterotomy (ES) followed by LC in fit patients (American Society of Anesthesiologists (ASA) grades I and II) has proven the two approaches to be equally effective but with a shorter hospital stay after the single-stage approach . HoweverThe patients presenting gallbladder stones and secondary CBD stones were treated according to the authors' previously published algorithm and surgical technique  20]. St. St20]. Out of 121 cases, 61 patients who were elderly at the time of surgery had passed away for unrelated causes but were declared free from biliary symptoms from their relatives. Fourteen patients (11.5%) were lost to follow-up. The 46 remaining patients from the original series underwent the follow-up protocol , with a medium follow-up of 17.1 years (range 12.6–22.7 years).γ-glutamyl transpeptidase, and transaminases together with leukocytosis. One of the two patients reporting dyspepsia had moderately increased transaminases levels but no biochemical signs of bile stasis. US evaluation was performed in every patient (100%) while CMRI was obtained in symptomatic patients only. No stones or biliary sludge was observed at US in 45 patients (97.83%). In the only symptomatic patient no ductal stones were seen at US. This patient underwent CMRI that showed the presence of two stones measuring 1 and 0.8 cm, respectively, located in the distal common bile duct. ERCP with sphincterotomy was performed, with stones' removal. Neither signs of papillitis nor of ductal stricture at any level of the extrahepatic bile ducts were observed. In this case a mild increase in pancreatic enzymes occurred on the first day after ERCP-ES but subsequently resolved and the patient was discharged on postprocedural day III, after normalization of biochemical parameters. Follow-up data are summarized in Specific symptoms of bile stasis occurred in one (2.1%) female patient presenting with episodes of cholangitis that occurred sixteen years after LC + LCBDE. Two more (4.3%) male patients reported dyspepsia. Biochemistry was negative in all patients except for the patient with cholangitis who showed increased levels of alkaline phosphatase, Aim of this study is to report the longer-term results with a medium follow-up of 17 years in a consecutive series of unselected patients who underwent laparoscopic choledochotomy with T-tube biliary drainage. Complete follow-up data, including physical examination, laboratory exams, and imaging data, were obtained in the 46 patients who were available at the time of the follow-up call. Sixty-one patients had passed away for other reasons since many of them were already older than 65 years of age at the time of surgery . To the Single-stage laparoscopic treatment of gallstones and CBD stones has been adopted by few dedicated centers , 22–24 bIn the authors' opinion, laparoscopic transcystic duct exploration should be the technique of choice because it is less invasive. Laparoscopic choledochotomy should be reserved to patients in whom transcystic duct exploration is not possible or when intraoperative cholangiography shows the presence of unfavorable conditions for a transcystic duct approach. The ideal indications for the transcystic duct approach have been clearly defined: (a) a dilated cystic duct, joining the CBD on its lateral side; (b) a limited number of ductal stones (<4); (c) small size of ductal stones located in the CBD , 16, 42.p = 0,037) following longitudinal choledochotomy at long-term follow-up (8–10 years) [Patients who undergo a choledochotomy are generally considered to be at greater risk of long-term morbidity, namely, stricture, also up to twenty years after surgery as reported after open surgery . The mai0 years) . per se does not prevent the occurrence of bile leaks, which has been reported when the drainage is in situ as well as after its removal [In the authors' series, a T-tube drainage was employed routinely after laparoscopic transverse choledochotomy because the aim was to evaluate the safety and efficacy of this (by that time) new procedure, particularly in terms of residual stones' rate. Later, the indications for T-tube placement were reviewed and now it is employed selectively only in case of intraoperative instrumental manipulation of the papilla, such as transpapillary passage of basket or choledochoscope. Several other complications related to the use of T-tube biliary drainage are reported in the literature and include fluid electrolyte imbalance, sepsis, premature dislodgement, bile leak, biliary peritonitis, prolonged biliary fistula, and late biliary stricture , 52. Som removal , 52. A C removal  comparedThe main criticisms against laparoscopic single-stage treatment of gallstones and CBD stones are related to purported higher costs, the need for advanced laparoscopic biliary expertise, and the fact that the operative time for LC with LCBDE may not be foreseen in every case. The cost of LC with LCBDE has been reported by several authors to be lower than for the two-stage approach –56. ConcIn conclusion, in the authors' opinion LCDBE during LC should be the treatment of choice in elective patients with gallstones and CBD stones in centers where adequate expertise and a dedicated instrumentation are available. This procedure should be performed by a laparoscopic transcystic duct approach whenever possible and by transverse choledochotomy in selected cases. Laparoscopic transverse choledochotomy during LC has proven to be safe and effective also at longer-term follow-up, with no evidence of common bile duct stricture and with a stones' recurrence rate that is much lower than that reported in the literature after endoscopic sphincterotomy. For good results to be achieved, the choledochotomy must be performed with a correct surgical technique aimed at preventing the occurrence of ischemia, with a T-tube of small diameter, which is left in place long enough for a mature sinus tract to develop, so as to avoid the occurrence of bile peritonitis after biliary drainage removal.


Bacillus subtilis contains two glutamate dehydro genase-encoding genes, rocG and gudB. While the rocG gene encodes the functional GDH, the gudB gene is cryptic (CRgudB) in the laboratory strain 168 due to a perfect 18 bp-long direct repeat that renders the GudB enzyme inactive and unstable. Although constitutively expressed the GudBCR protein can hardly be detected in B. subtilis as it is rapidly degraded within stationary growth phase. Its high instability qualifies GudBCR as a model substrate for studying protein turnover in B. subtilis. Recently, we have developed a visual screen to monitor the GudBCR stability in the cell using a GFP-GudBCR fusion. Using fluorescent microscopy we found that the GFP protein is simultaneously degraded together with GudBCR. This allows us to analyze the stability of GudBCR in living cells. By combining the visual screen with a transposon mutagenesis approach we looked for mutants that show an increased fluorescence signal compared to the wild type indicating a stabilized GFP-GudBCR fusion. We observed, that disruption of the arginine kinase encoding gene mcsB upon transposon insertion leads to increased amounts of the GFP-GudBCR fusion in this mutant. Deletion of the cognate arginine phosphatase YwlE in contrast results in reduced levels of the GFP-GudBCR fusion. Recently, it was shown that the kinase McsB is involved in phosphorylation of GudBCR on arginine residues. Here we show that selected arginine-lysine point mutations of GudBCR exhibit no influence on degradation. The activity of McsB and YwlE, however, are crucial for the activation and inhibition, respectively, of a proteolytic machinery that efficiently degrades the unstable GudBCR protein in B. subtilis.The Gram-positive model bacterium  Posttranslational modifications of proteins allow bacteria to control several important cellular processes. Phosphorylation is such a posttranslational modification event that can severely affect the function of a protein, which is targeted by a specific kinase . In bactBacillus subtilis is controlled by reversible phosphorylation of a tyrosine residue  RocG and GudB, respectively  due to a perfect 18 bp-long direct repeat (DR). This occurs in the part of the gene encoding the active center of the enzyme .We are interested in the regulation of glutamate metabolism in ectively . Some isonal GDH . In thise enzyme . The Gudry phase , 2013. Atability , other fresidues . It is tCR fusion to monitor the GudBCR stability in vivo. By applying microscopical and biochemical techniques, we found that GFP and GudBCR are simultaneously degraded. Thus, the visual screen is suitable to analyze the cellular amount of GudBCR. To identify novel factors that are involved in GudBCR degradation, we combined the visual screen with a transposon mutagenesis approach. Afterward we looked for mutants that show an increased fluorescence, indicating increased amounts of the GFP-GudBCR fusion. Among the transposants we found one insertion in the mcsB gene encoding the arginine kinase McsB. Moreover, inactivation of the cognate phosphatase YwlE resulted in a decreased fluorescence of a strain synthesizing the GFP-GudBCR fusion. The possible mechanisms of how the activity of the kinase McsB and the cognate phosphatase YwlE affect the amount of the GudBCR protein are discussed.In the present study, we apply a visual screen that is based on a GFP-GudBTable 1. B. subtilis chromosomal DNA was isolated using the DNeasy Blood & Tissue Kit . Plasmids were isolated from Escherichia coli using the Nucleospin Extract Kit . PCR products were purified using the PCR Purification Kit . Phusion DNA polymerase, restriction enzymes and T4 DNA ligase were purchased from Thermo Scientific (Germany) and used according to the manufacturer’s instructions. Other chemicals and media were purchased from Sigma-Aldrich (Germany), Carl Roth  and Becton Dickinson . Sequencing of DNA was performed by the SeqLab Sequence Laboratories .The oligonucleotides were purchased from Sigma-Aldrich (Germany) and are listed in B. subtilis strains (Table 2) were grown in LB and SP medium, respectively. LB and SP plates were prepared by the addition of 17 g agar/l  to LB and SP , respectively. When required, media were supplemented with antibiotics at the following concentrations: ampicillin (100 μg/ml), kanamycin (10 μg/ml), chloramphenicol (5 μg/ml), lincomycin/erythromycin (25/2 μg/ml), tetracyclin (12.5 μg/ml), and spectinomycin (150 μg/ml). B. subtilis was transformed with plasmid and chromosomal DNA according to a previously described two-step protocol . The PCR products were digested with the enzymes BamHI and PstI and ligated to the plasmid pBQ200 that was cut with the same enzymes. The plasmids harboring the ywlE and mcsB genes and their native ribosome-binding sites were designated pBP183 and pBP186, respectively. Expression of the genes is driven by the constitutively active degQP promoter (CRgfp-gudB mutant (designated as CRgfp-gudB-mut), encoding the GudBCR protein in which the arginine residues 56, 83, 421, and 423 were replaced by lysine, was constructed by the Multiple-mutation reaction . The insertion sites of the mini-Tn10 transposon were determined by DNA sequencing of the plasmids using the oligonucleotides pIC333_seq up and pIC333_seq down.For transposon mutagenesis of the r pIC333  as descrr pIC333 . The traoli DH5α . For allFor Western blot analyses, proteins present in 20–50 μg of cell free crude extracts were separated by 12.5% SDS PAGE and transferred onto polyvinylidene difluoride membrane  by semi-dry electroblotting. Anti-GFP , anti-YwlE, anti-McsB, and anti-GapA polyclonal antibodies were diluted 1:10.000, 1:1000, 1:5.000, and 1:30.000, respectively, and served as primary antibodies. The antibodies were visualized using anti-rabbit immunoglobulin alkaline phosphatase secondary antibodies  and the CDP-Star detection system  as described previously .B. subtilis colonies were taken with a stereo fluorescence microscope Lumar.V12  equipped with the ZEN lite 2011 (blue edition) software. The applied filter set was Lumar 38 for eGFP detection . Images were taken at room temperature and an exposure time of 1 s.For fluorescence microscopy, cells were grown in SP medium to optical densities as indicated, and analyzed on agarose microscopy slides. Fluorescence images were obtained with an Axioskop 40 FL fluorescence microscope, equipped with digital camera AxioCam MRm and AxioVision Rel (version 4.8) software for image processing  and Neofluar series objective at x 100 primary magnification. The applied filter set was eGFP HC-Filterset  for GFP detection. Pictures of CR fusion protein were determined by monitoring the fluorescence  in a growing bacterial culture using the Synergy MX II multimode microplate reader (BioTek). For this purpose, 4 ml LB medium were inoculated with the precultures to an OD600 of 0.1. The cultures, that had an approximate OD600 of 1.0, were used to inoculate a 96 well plate  containing 180 μl medium per well. To avoid evaporation, the outermost wells were filled with 200 μl sterile water. The plates were incubated for a maximum of 10 h at 37°C and fast shaking speed. OD600 was measured every 10 min throughout the experiment. Background fluorescence of the parental strains was subtracted from the raw fluorescence of all gfp fusion strains at the same OD600. The cellular amounts of the GFP-GudBCR fusion protein correspond to the fluorescence divided by the OD600 at each time point.Cellular amounts of the GFP-GudBCR protein is degraded  and BP26 (gfp-gudB) were grown in liquid medium, mixed in a 1:1 ratio and appropriate dilutions were propagated on SP plates to allow growth of individual colonies. By visual inspection of the plates using a stereo fluorescence microscope we found several colonies that were grown close to each other and showed different fluorescence signals . We then re-streaked some of the colonies showing different fluorescence signals on agar plates to obtain individual colonies. Next, we performed colony PCRs and confirmed that the higher and lower fluorescence signals were due to the presence of the gfp-gudB and CRgfp-gudB alleles, respectively. In conclusion, the visual screen seems to be suitable to look for mutants, lacking factors that enhance or decrease proteolytic degradation of GFP-GudBCR.The fact that also the GFP-GudBdegraded , 2013 quy stable  and syntCR, we performed a transposon mutagenesis with strain BP25 (CRgfp-gudB) using the mini-Tn10 delivery vector pIC333 . Appropriate dilutions of the transposants were propagated on SP plates that were incubated for 48 h at 42°C. By visual inspection of about 8000 transposants we could identify one mutant that showed no fluorescence signal, whereas a second mutant showed an increase in fluorescence intensity. While the first mutant had obviously lost the ability to synthesize GFP because the transposon was inserted into the gfp gene, the mutant showing increased fluorescence had integrated the transposon at position 580 into the arginine kinase encoding mcsB gene . The lack of McsB also resulted in the formation of large aggregates of the GFP-GudBCR fusion protein at the cell poles , an observation that can be made when aggregation prone proteins are synthesized in bacteria . In case the arginine phosphatase YwlE counteracts the function of its cognate kinase McsB, we expected to observe that single cells as well as colonies of the ywlE mutant BP74 would show a reduced fluorescence. This was indeed the case for single cells of the ywlE mutant strain in comparison to cells of the mcsB mutant and parent strains BP69 and BP25, respectively . Although less pronounced, fluorescence of the ywlE mutant was also reduced at the level of single colonies . However, a quantification of the fluorescence of the GFP-GudBCR fusion protein monitored in growing cultures in the ywlE mutant strain clearly demonstrates that the phosphatase YwlE affects GudBCR stability .To underpin the role of arginine phosphorylation in the degradation of the GudBCR fusion protein. For this purpose, we cultivated the parent strain BP25 as well as the mcsB and ywlE mutant strains BP69 and BP74, respectively, in SP medium until stationary phase (the OD600 was around 3.0) and analyzed the amounts of the GFP-GudBCR fusion protein by Western blotting using GFP-specific antibodies. As shown in Figure 2C, in strain BP69 lacking McsB the cellular amount of the GFP-GudBCR fusion protein was strongly increased. By contrast, the inactivation of the ywlE gene resulted in a decrease of GFP-GudBCR levels. In conclusion, the semi-quantitative Western Blot analyses are in perfect agreement with the fluorescence microscopical studies.Next, we confirmed that the kinase McsB and the phosphatase YwlE affect the cellular levels of the GFP-GudBgfp-gudB) synthesizing the active GFP-GudB fusion and the isogenic mcsB and ywlE mutant strains BP311 and BP75 (Table 2), respectively, in SP medium until stationary phase (OD600 of about 3.0). Afterward, we quantified the amount of the GFP-GudB protein by Western blotting using antibodies specific for GFP. As shown in Figure 2C, irrespective of the absence of either McsB or YwlE all strains synthesized similar amounts of the active GFP-GudB fusion protein. In conclusion, only the cellular amount of the inactive GFP-GudBCR but not that of the active GFP-GudB fusion protein is significantly affected by McsB.Subsequently, we wanted to answer the question of whether McsB and YwlE do also influence the cellular amounts of the enzymatically active GFP-GudB fusion protein lacking the duplication of three amino acids in the active center of the enzyme. For this purpose, we cultivated the parent strain BP26  and pBP183 (ywlE). Both plasmids are derivatives of the non-integrative overexpression plasmid pBQ200 and gene expression is driven by the constitutively active degQP promoter  served as a control. As shown in Figure 3A, the emitted fluorescence of all cultures was similar during exponentially growth. In the stationary phase the fluorescence signal was much higher in the mcsB mutant strain BP69 when compared to that of the parent strain BP25. By contrast, inactivation of the ywlE resulted in a strong decrease of the fluorescence signal. Overexpression of the mcsB and ywlE genes in the mcsB and ywlE mutant strains BP69 and BP74, respectively, restored the fluorescence signal in the stationary phase almost to the extent of the parent strain. Western blot experiments using antibodies specific for McsB and YwlE confirmed overexpression of the arginine kinase and the phosphatase from the complementation plasmids in the mcsB and ywlE mutant strains BP69 and BP74, respectively . In conclusion, the cultivation experiments to detect the cellular levels of the GFP-GudBCR fusion protein are in good agreement with the previous experiments showing that the lack of the McsB and YwlE results in elevated and reduced levels, respectively, of the inactive GDH. Moreover, together with the Western blot experiments the cultivation experiments also revealed that the mcsB and ywlE mutations can be complemented by expressing the mcsB and ywlE genes from plasmids.For complementation studies of the promoter . The plamcsB gene lies immediately upstream of the clpC gene in the ctsR mcsA mcsB clpC operon. Since the mcsB mutation can be complemented, it can be ruled out that enhanced cellular levels of the GFP-GudBCR fusion are a consequence of a polar effect of the transposon insertion into the mcsB gene leading to a reduced clpC expression and a lower proteolytic activity. However, the lower proteolytic activity in the mcsB mutant strain might be due to the missing of McsB-dependent activation of the ClpC-ClpP protease complex. To exclude this possibility, we compared the cellular amounts of the GFP-GudBCR fusion in the background of the clpC and clpP mutant strains BP98 and BP99, respectively, to that of the parent strain BP25 (CRgfp-gudB). For this purpose, we grew the bacteria in SP medium and collected samples from exponential and stationary phases and performed Western blot analyses . The strain BP26 (gfp-gudB) as well as the mcsB and ywlE mutant strains BP69 and BP74, respectively, served as controls. As expected, in contrast to the inactive GFP-GudBCR fusion protein the active GFP-GudB variant was more abundant during exponential and stationary phase. Moreover, as observed in the previous experiments, the GFP-GudBCR levels were increased and decreased in the mcsB and ywlE mutants, respectively . Finally, the GFP-GudBCR levels in the clpC and clpP mutant strains BP98 and BP99, respectively, were similar to that of the parent strain BP25 (CRgfp-gudB). Using GapA and GFP antibodies, we show that only the GFP-GudBCR fusion but not GapA was degraded in stationary growth phase samples. Thus, McsB is involved in GudBCR degradation in a rather ClpP and ClpC-independent manner.The CR protein is phosphorylated on the arginine residues at positions 56, 83, 421, and 423 , the mcsB mutant strain BP69 (mcsBgfp-gudBCR), the quadruple GFP-GudBCR mutant strain BP230 (gfp-gudBCR-mut (R56K R83K R421K R423K)), and the isogenic mcsB mutant strain BP231 (mcsBgfp-gudBCR-mut (R56K R83K R421K R423K)) were cultivated in SP medium. Simultaneously, the cellular levels of the fusion proteins were determined by monitoring the fluorescence during bacterial growth. As shown in Figure 5, the fluorescence measurements revealed that the cellular levels of the fusion proteins in strains BP25 (CRgfp-gudB) and BP230 (CRgfp-gudB-mut (R56K R83K R421K R423K)) was much lower in comparison to those of the isogenic mcsB mutant strains BP69 (CRmcsB gfp-gudB), and BP231 (CRmcsB gfp-gudB-mut (R56K R83K R421K R423K)). In conclusion, these observations suggest that phosphorylation of the arginine residues 56, 83, 421, and 423 sites is rather not important for the degradation of the inactive GudBCR protein.In a recent phosphoproteome analysis it has been shown that the inactive GudB and 423 . To evalmcsB arginine kinase gene resulted in stabilization of the inactive GDH GudBCR during stationary growth phase of B. subtilis. Thus, beside its role in controlling the degradation of the DNA-binding transcription factor CtsR . Thus, the degradation of GudBCR seems to be rather indirectly influenced by McsB. Finally, an unknown proteolytic machinery that remains to be identified might be responsible for the degradation of the misfolded and inactive GDH GudBCR. On one hand the activity of the proteolytic machinery might be controlled by McsB-dependent phosphorylation of an unknown adaptor protein that specifically recognizes GudBCR and directs the protein to the protease for degradation. On the other hand McsB could be important for the activation of one of the AAA+ proteases or other unknown proteases that remain to be identified. One could also envision that McsB acts itself as the adaptor that mediates proteolysis of the GudBCR protein. The interaction between McsB and GudBCR could result in coincidental phosphorylation of the GDH. This could also be the case for the other arginine phosphorylations of the B. subtilis proteome  when exogenous glutamate is not available (CRgudB gene and synthesize in addition to RocG the functional GDH GudB. Moreover, the selective pressure acting on the B. subtilis strain 168 might be an explanation for the observation that the cryptic CRgudB gene is stably inherited since the bacterium has been domesticated. Recently, it has been shown that bacteria rapidly loose genes and reduce their genome sizes when adapted to specialized environments. This might also be observed in the laboratory by experimental evolution of bacterial cell populations (B. subtilis affords to waste energy by permanently synthesizing an inactive enzyme that is subject to rapid degradation. However, under certain growth conditions it must be advantageous for B. subtilis to harbor the cryptic CRgudB gene that, when activated by spontaneous mutagenesis (B. subtilis 168 expressing rocG and gudB can use glutamate as a singly source of carbon and nitrogen (As described above it is interesting to note that only the domesticated unstable . It has lutamate . This hyvailable . Obviousulations . Therefoagenesis , encodesnitrogen . Thus, uCR. This goal might be achieved by monitoring the cellular amounts of the GFP-GudBCR fusion protein in a mutant collection that have inactivated all non-essential genes by targeted gene deletion or by a next time saturated transposon mutagenesis. The identification of novel factors that are involved in GFP-GudBCR proteolysis might be facilitated by monitoring growth and fluorescence over time because the fusion protein seems to be preferentially degraded in stationary phase. Moreover, it will be interesting to address the question whether arginine phosphorylation influences the physiological functions of other proteins in B. subtilis.In the future it will be interesting to identify additional factors that are involved in the rapid degradation of the enzymatically inactive GDH GudBThe 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.


Different types of carbohydrates have diverse glycemic response, thus glycemic index (GI) and glycemic load (GL) are used to assess this variation. The impact of dietary GI and GL in all-cause mortality is unknown. The objective of this study was to estimate the association between dietary GI and GL and risk of all-cause mortality in the PREDIMED study.The PREDIMED study is a randomized nutritional intervention trial for primary cardiovascular prevention based on community-dwelling men and women at high risk of cardiovascular disease. Dietary information was collected at baseline and yearly using a validated 137-item food frequency questionnaire (FFQ). We assigned GI values of each item by a 5-step methodology, using the International Tables of GI and GL Values. Deaths were ascertained through contact with families and general practitioners, review of medical records and consultation of the National Death Index. Cox regression models were used to estimate multivariable-adjusted hazard ratios (HR) and their 95% CI for mortality, according to quartiles of energy-adjusted dietary GI/GL. To assess repeated measures of exposure, we updated GI and GL intakes from the yearly FFQs and used Cox models with time-dependent exposures.We followed 3,583 non-diabetic subjects . As compared to participants in the lowest quartile of baseline dietary GI, those in the highest quartile showed an increased risk of all-cause mortality . In the repeated-measures analyses using as exposure the yearly updated information on GI, we observed a similar association. Dietary GL was associated with all-cause mortality only when subjects were younger than 75 years.High dietary GI was positively associated with all-cause mortality in elderly population at high cardiovascular risk. The epidemiological transition and increased prevalence of chronic-degenerative diseases in the elderly population is related to nutritional status. In Spain, 35% of the population aged 65 years or more is obese Currently, dietary recommendations from FAO and WHO to prevent chronic diseases are aimed to reduce or change the proportion of fats to a healthier profile, while increasing the carbohydrate content of the diet Current research has evaluated the relationship between dietary GI and GL in the prevention and study of obesity, CVD, cancer and many other health problems, such as macular degeneration The literature to confirm the relationship between GI and GL for overall fatal events is scarce. In fact, usually, references are related to specific causes of fatal events such as cancer The association between GI/GL and all-cause mortality is still unclear; therefore, we aimed to evaluate the association of the quality of carbohydrates on all-cause mortality in an elderly population at high cardiovascular risk.This study was conducted within the frame of the PREDIMED study (PREvención con DIeta MEDiterránea), a randomized, multicenter, parallel group, single-blinded dietary intervention trial conducted in Spain  2), or family history of premature CVD).The PREDIMED study included 7447 participants, aged between 55 and 80 years. Subjects without prior CVD were selected when they met at least one of the following criteria: presence of type 2 Diabetes Mellitus (T2DM) or the presence of three or more cardiovascular risk factors , low HDL cholesterol (≤40 mg/dL in men and ≤50 mg/dL in women), overweight or obesity . Exclusion criteria were: subjects without follow-up (n = 125), with values of total energy intake outside of predefined limits  (n = 86), use of anti-diabetic medication (n = 21) and incomplete data in any variable used in the analyses (n = 18). Overall, 3,583 subjects were analyzed in this study. The rationale for exclusion of persons with a baseline history of diabetes mellitus  was potential effect modification and potential changes in the diet as a result of diagnosis and treatment of T2DM.Trained dietitians used a 137–item food frequency questionnaire (FFQ) to assess dietary habits by face-to-face interviews. This FFQ was repeatedly administered each year during follow-up. The FFQ has been validated in a sample of subjects with similar characteristics to the participants of the PREDIMED study We assigned the GI of each food of the FFQ using Louie et al. protocol After the GI assignment, we estimated dietary GI for each individual summing the GI of each food multiplied by the amount of available carbohydrate consumed (g/day) and divided by the total available carbohydrate amount. For dietary GL, the sum of the GI multiplied by the amount of available carbohydrate consumed was divided by 100 2) squared. We estimated energy expenditure using a validated Spanish version of the Minnesota leisure-time physical activity questionnaire Socio-demographic information, medical history and lifestyle habits were collected via specific questionnaires carried out by trained personnel. Height and weight were measured wearing light clothes, barefoot, using a wall-mounted stadiometer and calibrated scales. BMI was estimated as weight (kg) divided by the height . Different Cox regression models were used to estimate multivariable-adjusted hazard ratios (HR) and their 95% CI for all-cause mortality according to baseline quartiles of energy adjusted dietary GI and GL. A first model was adjusted for sex, age (years), recruitment center and intervention group . A second model was further adjusted for potential confounders including: smoking , education , marital status , physical activity (continuous), BMI (continuous), self-reported history of cancer , self-reported history of arterial hypertension , self-reported history of dyslipidemia , self-reported history of cardiovascular disease , total energy intake (continuous), alcohol intake (continuous) and dietary fiber intake . The third model was further adjusted for saturated fatty acids  and monounsaturated fatty acids . In all the analyses, recruitment center was entered into the model through the strata statement. Tests of linear trend across increasing quartiles of GI and GL were conducted by assigning the medians to each quartile and these variables were treated as continuous in the multivariate models. To assess a possible interaction between dietary GI/GL and sex or intervention group, we introduced product terms in the different multivariable models and considered p<0.05 in the likelihood ratio test as statistically significant.To update GI and GL intake with all available longitudinal data, we used Cox regression models with time-dependent exposures. Three models were adjusted using the same covariates used for the models of baseline analysis. For dietary measures, we used the cumulative average of food intakes from baseline to the censoring events. BMI and physical activity were also yearly updated. Because changes in diet after development of diabetes may confound the association between exposure and mortality 2) and including only obese subjects, excluding participants with more than 6 years of follow-up and exclusion of subjects aged 75 years or more. Statistical analyses were performed using Stata 12.0  and the significance level was set at p<0.05.Finally, sensitivity analyses were conducted using quartiles of energy-adjusted GI and GL specific for different groups of the population to observe the effect of changing energy limits , including population with T2DM, excluding obese subjects (BMI≥30 kg/m2), higher consumption of alcohol and carbohydrate but less intake of protein, fat and dietary fiber than the subjects in the bottom quartile of dietary GI. Characteristics according to dietary GL were similar to GI excepting that subjects in the highest quartile of GL were more often female, older, less likely to be current smokers, more likely to have only primary education, less likely to be married and physically active, had lower alcohol consumption, lower energy intake had higher intake of fiber when compared to the lowest quartile. The proportion of prevalent diseases such as cancer or arterial hypertension was similar across quartiles of dietary GI and GL. According to intervention group, mean dietary GI and GL were significantly higher when comparing the highest quartiles vs. the lowest.In this study, participants were followed for a median of 4.7 years. Of 3,583 subjects, 123 cases of all-cause mortality were registered. The mean (SD) dietary GI and GL of participants at recruitment was 57.6 (4.7) and 117.6 (24.1) respectively. We found a significant association between the highest quartile of dietary GI and all-cause mortality Table 2.Results from sensitivity analyses comparing the risk of mortality between the upper quartiles (quartile 3 and quartile 4) of GI and GL with reference to the lowest quartile are shown in We found that higher intake of baseline dietary GI was associated with an increased risk of all-cause mortality in 3,583 non-diabetic elderly subjects. This association remained significant in the repeated measurement analysis. With respect of dietary GL, we observed a higher risk of all-cause mortality only in subjects at high risk of CVD younger than 75 years.Our results differ from a cohort of Swedish men, aged from 45 to 79 years with prior cardiovascular disease, where no association was found between dietary GI nor GL and all-cause mortality Our results also agree with a previous study where Oba et al. reported an association of similar magnitude between dietary GI and mortality from stroke risk in women. No associations were observed between dietary GL and the risk of total stroke or death from ischemic stroke We identified an association between the highest quartiles of dietary GI and risk of all-cause mortality in obese subjects. Two recent meta-analyses have shown that the effect of dietary GL on the risk of coronary heart disease appeared more evident in subjects with higher BMI, although the authors recommended treating this information with caution because of the limited evidence and diversity among the BMI cut-off points used across studies Furthermore, higher values of dietary GI or GL could increase the risk of all-cause mortality by raising the risk of chronic diseases. Recent meta-analyses have shown a relationship between dietary GI, GL and increased risk of CVD in women but not in men After a high-GI meal, blood glucose concentration increases at least twice that after a low-Gl meal with the same nutrients and energy. This hyperglycemia stimulates insulin release and inhibits glucagon liberation. High insulin-to-glucagon ratio affects normal anabolic responses such as uptake of nutrients by insulin-responsive tissues, glycogenesis, lipogenesis, and inhibits gluconeogenesis and lipolysis. From 2 to 4 hours after a high-GI meal, the absorption from the gastrointestinal tract declines, but the effects of hiperinsulinemia and low glucagon remain. Blood glucose drops to lower hypoglycemic range and release of free fatty acid is more suppressed than compared with a low-GI meal. In the late postprandial period, the low circulating concentration of metabolic fuel activates the hormone response that restores euglicemia and elevates free fatty acids to higher levels than observed after low-GI meals Eventually these postprandial responses may contribute to insulin resistance and obesity Additionally, higher dietary GI has been associated with small increases in C-reactive protein A study conducted in healthy elderly Europeans found that a posteriori plant-based dietary pattern was associated with lower all-cause mortality. This plant-based pattern with high intakes of vegetables, vegetable oils, fruit, legumes and pasta/rice/other grains and low intakes of potatoes, margarine and non-alcoholic beverages, was correlated with the MeDiet pattern The limitations of this study are mainly methodological. Firstly, due to the scarcity of GI values for Spanish foods, we used as reference GI data from other countries. This fact could be a source of bias, because GI values may differ wide ranges, depending on variety, processing and cooking In summary, this study provides evidence that high GI diets are related to increased risk of all-cause mortality in non-diabetic elderly subjects at high cardiovascular risk. In the sensitivity analyses this association was statistically significant when subjects were younger than 75 years or obese. Nevertheless, more evidence is necessary to evaluate these associations in different population groups and further studies that elucidate the mechanisms supporting the inclusion of GI and GL in dietary recommendations.