In six trials, orally administered vitamin C in doses of 1–3 g/day (weighted mean 2.0 g/day) reduced the length of ICU stay by 8.6% (p = 0.003). In three trials in which patients needed mechanical ventilation for over 24 hours, vitamin C shortened the duration of mechanical ventilation by 18.2% . Given the insignificant cost of vitamin C, even an 8% reduction in ICU stay is worth exploring. The effects of vitamin C on ICU patients should be investigated in more detail.A number of controlled trials have previously found that in some contexts, vitamin C can have beneficial effects on blood pressure, infections, bronchoconstriction, atrial fibrillation, and acute kidney injury. However, the practical significance of these effects is not clear. The purpose of this meta-analysis was to evaluate whether vitamin C has an effect on the practical outcomes: length of stay in the intensive care unit (ICU) and duration of mechanical ventilation. We identified 18 relevant controlled trials with a total of 2004 patients, 13 of which investigated patients undergoing elective cardiac surgery. We carried out the meta-analysis using the inverse variance, fixed effect options, using the ratio of means scale. In 12 trials with 1766 patients, vitamin C reduced the length of ICU stay on average by 7.8% (95% CI: 4.2% to 11.2%;  For many centuries, scurvy was a severe disease most commonly afflicting sailors on long voyages ,14,15,16In controlled trials, vitamin C has improved endothelial function ,19,20, lIn experimental vitamin C deficiency studies, scurvy is induced when dietary vitamin C intake and plasma vitamin C levels are below approximately 10 mg/day and 11 μmol/L, respectively ,60,61,62While scurvy is relatively rare nowadays, it has not disappeared altogether. A number of case reports have described patients with overt scurvy in diverse hospital settings, including ICUs ,79,80,81A survey of elderly Scottish patients hospitalized because of acute respiratory infections reported that 35% of patients had vitamin C plasma levels less than 11 μmol/L . One stuGiven this, it would seem reasonable to screen plasma vitamin C levels in hospital patients when appropriate and administer vitamin C to patients with particularly low levels, irrespective of whether vitamin C is believed to be effective for conditions other than scurvy.Furthermore, there is evidence that vitamin C metabolism is changed in many conditions that involve physiological stress, such as infections, surgery, traumas, and burns, in which case vitamin C levels can decline dramatically ,100,101.Given that vitamin C has diverse effects on medical conditions ,47,48,49We included controlled trials that compared the length of ICU stay and/or the duration of mechanical ventilation between vitamin C and control groups. We included trials in which the administration of vitamin C was the only difference between the study groups, so that trials which administered other therapies as well as vitamin C were included, if the other therapies were the same for both trial groups. We did not limit our search to randomized trials and we did not require placebo control. We included all doses and all durations of vitamin C administration.We searched MEDLINE (Ovid), Scopus and the Cochrane Register  from their inception to 20 January 2019. We did not impose any restrictions on the language of publication. The search strategies are detailed in The primary outcome of interest in our study was length of ICU stay, and the secondary outcome was duration of mechanical ventilation. Moreover, we collected data for tertiary outcomes from the included trials when they appeared relevant for this review. These included multi-organ failure scores, inflammation markers, dose and duration of norepinephrine administration, creatine kinase MB isoenzyme (CK-MB) levels, requirement for fluid infusions, and cardiac index during follow-up (see Results).Both authors independently screened the titles and abstracts and identified trials for inclusion. One author (HH) extracted study characteristics and outcomes from the trials and entered the data in a spreadsheet. Both authors checked the data entered against the original trial reports.Both authors assessed the quality of included trials for the following criteria: random sequence generation, allocation concealment, blinding of patients and personnel, blinding of outcome assessment, and incomplete outcome data . Each quWe analyzed the duration data on the relative scale, since there is considerable evidence that the relative scale is usually more informative than the absolute scale for the analysis of continuous outcomes ,129,130.χ2 test and the I2 statistic to assess statistical heterogeneity among the trials in each meta-analysis [I2 greater than about 50% indicate moderate heterogeneity, and over 75% indicate a high level of heterogeneity. We used two-tailed p values in this analysis.We pooled the selected trials with the meta procedure of the meta program package of the R statistical software , using tanalysis . Values p-values, since, for skewed data such as the length of ICU stay, it is preferable to the t-test. In some trials, the MW p-values were incompatible with the reported SD values. Therefore, to keep our analyses consistent with the MW p-values, for five studies [p-value  test in their calculation of the  studies ,114,121,[p-value . For oneICU stay  . Of the trials that reported sex distribution, the proportion of males varied from 57% to 79% (median 70%) .Vitamin C was administered orally in seven trials and intravenously in 11 trials. All oral administration trials investigated cardiac patients and the dose ranged from 1 to 3 g/day. The 11 intravenous administration trials include all five non-cardiac studies and the dose ranged from 0.5 to 110 g/day. Three trials administered vitamin C on one single day only, and 11 administered vitamin C for four days or longer .There was substantial variation in patient selection between the trials . The carThe majority of the trials were randomized . Two triThe risk of bias assessment of the trials is shown in Four trials did not report the methods in sufficient detail to enable conclusions to be drawn about the possibility of bias caused by poor blinding ,114,118.Although plasma vitamin C levels at baseline and during treatment are highly informative characteristics of studies on vitamin C, only one trial reported plasma levels at the baseline. Fowler reported that at enrollment the mean plasma vitamin C level of their patients was 18 μmol/L . Plasma In our meta-analyses we pooled the results of the trials on the relative scale, calculating the ratio of means (RoM). For small effects, it is useful to transform the RoM values to a percentage scale. For example, RoM = 0.922 corresponds to a 7.8% mean decrease in the length of ICU stay.I2 = 90% (p = 10−24) (I2 = 58% (p = 0.002). In addition, when the Abdoulhossein (2018) trial with patients administered vitamin E [I2 = 35% (p = 0.088). Finally, three trials were very small [p-value, but moderately increased heterogeneity. We also excluded these three trials, and our final meta-analysis is shown in Over the 17 trials reporting on length of ICU stay, there is highly significant heterogeneity in the effect of vitamin C with = 10−24) . The sinitamin E  was alsory small ,115,117 p = 0.00003). There are substantial variations in the trials of the final meta-analysis, and moderate statistical heterogeneity over the 12 studies with I2 = 43%. Nevertheless, the confidence intervals of all the 12 trials are consistent with the pooled effect.In the final meta-analysis of 12 trials in We calculated the estimate of effect for selected subgroups . In a sep = 0.003). For these six trials, the weighted mean dose of vitamin C was 2.0 g/day. Thus, the effective dose of 2.0 g/day was associated with the 8.6% reduction in ICU stay.All of the six oral administration trials used vitamin C doses of 1 to 3 g/day. Overall, in these low dose oral administration studies, vitamin C reduced the length of ICU stay by 8.6% . In five trials with control group ICU stay from 3 to 5 days, corresponding to sicker patients, vitamin C reduced ICU stay by 10.1% (p = 0.0001). However, the confidence intervals for these groups were substantially overlapping . However, there was a significant difference in the effect of vitamin C on the duration of mechanical ventilation between those who were ventilated for less than 24 h and those who were ventilated for more than 24 h (p = 0.004). In meta-regression, the evidence for modification of the vitamin C effect by duration of mechanical ventilation was even stronger  (p = 0.7). In two trials with non-cardiac patients [p = 0.006).We identified six trials that reported the duration of mechanical ventilation in the vitamin C and control groups . On aver= 0.001) . In threpatients ,117, vitOur primary and secondary outcomes were the length of ICU stay and the duration of mechanical ventilation. Additionally, we collected data on other outcomes relevant in the ICU context; however, there are few and they are so heterogeneous that further meta-analyses were not feasible.Two small trials with sepsis patients were not informative about ICU stay and were excluded from Fowler  reportedp < 0.01 for both). Mortality over 28 days in the vitamin C group was reduced by RR = 0.22  based on 2/14 deaths in the vitamin C group and 9/14 in the placebo group.In another trial with sepsis patients, Zabet  reportedIn the earliest study, Dingchao  reportedp < 0.01). In addition, fluid retention was decreased by 68% (p < 0.01) and weight gain was 48% lower (p < 0.01) in the vitamin C group.Tanaka  reportedAbdoulhossein  reportedPoor healing of wounds and swelling of the gums are the most well-known symptoms of scurvy. At the biochemical level, they are explained by the role of vitamin C in collagen metabolism. However, vitamin C has a number of biochemical roles unrelated to collagen metabolism. For example, it participates in the synthesis of norepinephrine, which is a neurotransmitter, carnitine, which is involved in energy metabolism, and nitric oxide (NO) ,138,139.Vitamin C also participates in the demethylation of DNA and histones and thereby influences the epigenome ,146,147.The numerous biochemical effects of vitamin C can explain the large variety of symptoms that have been reported in experimental vitamin C deficiency studies, such as depression, anxiety, irritability, confusion, shortness of breath, fatigue, oliguria with edema especially of the lower extremities, anemia, bruising, hemorrhages, joint pains, joint effusions, peripheral neuropathy, and common colds that last longer than usual ,60,61,62According to case reports of scurvy, in addition to fatigue, anemia, bruising, hematomas, and different types of pain, vitamin C deficiency has been associated with various cardiovascular symptoms ,74,75,76Logically, low systemic vitamin C levels should be considered in the differential diagnosis of these symptoms observed in experimental vitamin C deficiency studies and in case reports of scurvy. Scurvy is a clinical diagnosis and not a diagnosis based on plasma vitamin C level. For example, Hodges concluded that “the subjects … had plasma values well above the deficiency level throughout the time they had frank scurvy. Furthermore, a comparison between plasma levels of ascorbate and pool sizes showed a very poor correlation… In general, it is fair to say that scurvy appeared when the body pool fell below 300 mg” In the 1930s, after vitamin C had been identified, there were reports stating that plasma vitamin C levels of hospital patients were much lower than those of healthy people ,187,188.At that time, the concept of oxidative stress was unknown, as were many biochemical and physiological functions of vitamin C that were identified much later. Thus, the biochemical concepts required to consider the potential physiological harms of very low vitamin C levels were not developed. Randomized trial methodology had also not evolved, to enable an unbiased scientific investigation of whether vitamin C administration actually benefits hospital patients with very low vitamin C levels. A low vitamin C level does not necessarily mean that administration of vitamin C would be beneficial. A particular vitamin C level may simply be a marker for the various pathological processes rather than being implicated in the actual processes themselves. If this was the case, there would be no benefit from administration of vitamin C. Nevertheless, low levels of vitamin C in hospital patients still occur ,87,88,89Before vitamin C was identified, scurvy was associated with an elevated risk of pneumonia ,48, whicA Russian study reported a linear dose response over two vitamin C doses on the length of hospital stay for pneumonia patients ,198. A SThe leap from the common cold to critical care is vast; however, the ubiquity and mild character of the common cold has led to a large number of studies of vitamin C for prevention and treatment being undertaken. These studies include a total of more than 10,000 participants and such a large data set allows for informative subgroup analyses and other comparisons ,41,42. TFirstly, the common cold leads to a decline in vitamin C levels in white blood cells, and while 0.2 g/day of vitamin C did not return the levels to normal, administration of 6 g/day did . Thus, iSecondly, there is evidence that the effect of vitamin C supplementation on common cold incidence is context dependent. Context-dependency means that we should not expect a uniform effect in all populations. Although vitamin C has no effect on the incidence of colds in the normal population, it halved the incidence of colds in participants undergoing heavy physical activity ,43,202, Thirdly, two common cold studies compared two different vitamin C doses, and both found linear dose-dependency ,42,204: Fourthly, some common cold studies have indicated that the effects of vitamin C may be greater for outcomes that reflect greater severity . In two Finally, a few common cold studies have indicated that in some contexts vitamin C administration may lead to greater effects in males than in females ,211,212.A meta-analysis found that vitamin C reduced post-operative atrial fibrillation (POAF) in trials conducted outside of the USA, but not in studies carried out in the USA, indicating context dependency of the effect . For sevIn patients undergoing cardiac surgery, vitamin C has increased cardiac perfusion after the operation  and decrIn one of the experimental scurvy studies, two participants developed acute cardiac complications, and a third developed shortness of breath and pain in the chest. Therefore, the trial was terminated for each of them, and all three showed striking improvements within a few days of receiving vitamin C . SeveralPneumonia is the most common cause for severe sepsis, accounting for about half of all cases . As descVitamin C has been beneficial in animal models of sepsis, for example, by decreasing mortality ,153,217.The highest vitamin C levels in the body are in the adrenal glands and brain . AdrenalOral and intravenous administration of vitamin C lead to substantially different plasma vitamin C levels. When 1.25 g of vitamin C was given intravenously, plasma peak level increased approximately six-fold compared with the level after oral administration of the same dose ,227. BecThere are a variety of biochemical reactions in which vitamin C plays a part, a large number of physiological processes that are consequently influenced, and a large number of clinical effects of vitamin C that have been observed in controlled trials ,47,48,49In our meta-analysis of 12 controlled trials with 1766 patients, we found that vitamin C shortened the duration of ICU stay on average by 8% . Meta-anWe did not restrict our study to randomized and placebo-controlled trials. Although methodological details should be reported so that readers can draw their own conclusions about the validity of studies , there iIn empirical analyses, the placebo-effect has not been quantitatively meaningful when the outcomes were objective . In partMost of the included trials were randomized . Two stuThe calculation of the 8% average decrease in ICU stay is reasonable when testing the null hypothesis that vitamin C has no effect on ICU stay. However, that estimate cannot be generalized to all ICU contexts, since the patients treated in ICU are highly diverse. All except one of the 12 trials in our main meta-analysis on ICU stay were with patients undergoing elective cardiac surgery . HoweverWe did not find any meaningful differences between the effects of oral and intravenous vitamin C ; howeverThe effect of vitamin C administration on the duration of mechanical ventilation was significantly greater for ICU patients who were more severely ill . We alsoGiven that all people obtain vitamin C from their food, and there is great variation in vitamin C intake, it is surprising that only one of the included trials measured and reported baseline vitamin C levels. Therefore, the possible role of baseline vitamin C level as a variable defining the benefit of giving vitamin C could not be investigated.There are studies not included in our systematic review that are nevertheless informative on this topic. Two controlled trials with critical care patients did not report the length of ICU stay or duration of mechanical ventilation but reported other benefits from vitamin C ,242.A controlled trial carried out in Bangladesh reported that 1 g/day intravenous vitamin C decreased mortality for tetanus by 100% in 62 patients aged 1 to 12 years, and by 45% in 55 patients aged 13 years and more . The greA randomized trial of 84 patients with acute pancreatitis compared 1 g/day and 10 g/day intravenous vitamin C for five days . The higIn addition to trials with vitamin C alone, there are controlled trials of vitamin C given with other antioxidants. However, if there is a benefit from a set of several antioxidants, it is not possible to tell which one of them was effective, or whether an interaction between several antioxidants led to the benefit as in Furthermore, some before-after studies have reported benefits from vitamin C or from vitamin C together with other antioxidants. However, there can be various temporal changes and other confounders that could bias before-after studies. The concern regarding bias in observational studies is the reason that evidence-based medicine focuses on controlled trials. However, if the differences in a before-after comparison are very large, it is not easy to explain them by confounding. Some before-after studies were consistent with the notion that vitamin C may be beneficial for critically ill patients ,259,260.In the US nutritional recommendations, the ’tolerable upper intake level’ of adults for vitamin C was stated to be 2 g/day. However, the basis for this upper limit is the appearance of diarrhea  which isSeveral studies have shown that 4 g/day of vitamin C increases plasma levels of critically ill patients to the levels of the general population ,104,105.High doses of vitamin C, over 10 g/day, and in some cases even over 100 g/day, have been administered both orally and intravenously for patients with low frequency of adverse effects ,235,262.The rare cases of oxalate nephropathy caused by particularly high doses of vitamin C should not be used as a counterargument for raising very low vitamin C plasma levels of ICU patients to the range of normal healthy people with doses of about 4 g/day ,104,105.In 1975, Thomas Chalmers published a meta-analysis in which he pooled the findings of eight vitamin C and common cold trials and calculated that colds were 0.11 (SE 0.24) days shorter in the vitamin C groups . Chalmerp = 0.01) days reduction in the duration of colds [However, in 1995 the Chalmers review was shown to be seriously flawed. There were errors in the extraction of data, inconsistency in the selection of studies for the meta-analysis, and errors in the calculations ,265. If of colds . UnfortuAnother influential and frequently cited review by Dykes and Meier from 1975 on vitamin C and the common cold  was alsoThe problems with the two reviews and with the analysis of the randomized trial may be partly explained by the preconceptions of the authors. Furthermore, the very wide acceptance of the two reviews and the randomized trial in mainstream medicine, e.g., reflected by their usage in textbooks and nutritional recommendations etc. , suggestPrejudice against vitamin C is not limited to the common cold. Richards documented bias against vitamin C when she compared the attitudes and arguments of physicians to three putative cancer medicines: 5-fluorouracil, interferon, and vitamin C ,273,274.In an analysis discussing the preconceptions in academic medicine on micronutrient supplements, Goodwin and Tangum gave several examples to support the conclusion that there has been systematic bias against the concept that vitamins might be beneficial in levels higher than the minimum required to avoid classic deficiency diseases . In otheUsing vitamin C to prevent or treat diseases is often classified in the field of alternative medicine. However, such a classification does not mean that vitamin C is ineffective for all conditions other than scurvy .We found statistically highly significant evidence that vitamin C can shorten the length of ICU stay. We consider that our finding is a proof of concept, strongly encouraging further research, rather than justifying recommendations for change in practice. In further studies, the dose-response relationship should be carefully investigated, and oral and intravenous administration should be compared directly. Given that some common cold studies found the benefit of vitamin C to be greater in males than females, the effects of vitamin C in the ICU context should be compared between sexes. ICU patients are a highly heterogeneous group, and evidently, one estimate of vitamin C effect should not be expected to apply to all patient groups. Our analysis on mechanical ventilation indicated that the benefits of vitamin C may be greater for patients with more severe illness.Studies of arbitrary combinations of antioxidants teach us little, since they cannot be compared or pooled and drawing conclusions about a specific antioxidant is impossible. Instead, factorial trials such as the one shown in Vitamin C costs only pennies per gram, whereas one day in the ICU may cost thousands of dollars; therefore, an 8% decrease in ICU stay from the administration of 2 g/day of vitamin C warrants further research.


Specifically, the effects of the sizes of negatively charged MNPs on the resultant amyloid fibrillary mixture was investigated. Our results of AFM images indicated that the interaction between MNPs and the fibrils commences immediately after adding MNPs to the fibril solution, and the actions of such MNPs-doped fibrillary interplay, either integration or segmentation, is strongly dependent on the size and volume concentration of MNPs. In the cases of 5 nm and 20 nm particles of equivalent volume concentration, the adsorption and agglomeration of MNPs onto the fibrillary surfaces was observed, whereas, interestingly, MNPs with diameter size of 10 nm enables segmentation of the slender fibrils into debris when a proper implemented volume concentration was found, which signifies utter destruction of the amyloid fibrillary structure.Nanomedicine compounds containing nanoparticles, such as iron oxides and gold, have been demonstrated to be effective in promoting different magnitudes of interaction with amyloid  The AFM images are processed and analyzed using NanoScope software which is freely accessible.Deposition of a variety of misfolded proteins of amyloidal form in living organism induces detrimental cytotoxicity that lethally disrupts functions of cell-specific organs , consequ FTDP-17 . These d FTDP-17 ,7,8, and fibrils . To gain fibrils ,11. In a fibrils . Up unti origins ,15,16,17 origins ,19. Fromtoxicity . Likewis fibrils , and sim fibrils ,22. To mnegative . Althouggregates . Moreove effects . This reHen egg white lysozyme (HEWL)  was obtained from Sigma-Aldrich Chemical Company . All other chemicals were purchased from either Sigma or Fluka and were of analytical reagent grade. The protocol of preparing the solution of lysozyme amyloid fibrils (LAFs) and NP-incorporated fibrillary assembly is of the following. In brief, HEWL powder was dissolved in 0.2 M glycine-HCl with pH of 2.2 and 80 mM NaCl to obtain a final concentration of 5 mg/mL. The prepared solution was heated for 2 h at 65 °C in an enclosed bottle with constant stirring at the speed of 250 round per minutes (rpm), of which the final appearance delineated by AFM imaging is shown in 2O) with Fe concentration of 5 mg/mL containing 0.02% of NaN3 were purchased from Ocean Nano Tech Co. . The NPs were coated with a monolayer of amphiphilic polymer atop a monolayer of oleic acid, which are used for conjugation with the surface of biological elements and dispersion in the acqueous solution, and retention of the particle shape, respectively [The spherical carboxyl iron oxide NPs of an assorted sizes of 5 nm, 10 nm and 20 nm dispersed in deionized (DI) water  in air environment, and the AFM tip used throughout the entire experimental session was the antimony-doped etched silicon probe with aluminum thin film coated on its backside .The values of zeta-potentials for individual samples were measured using Zetasizer . The zeta-potential of MNPs solution was negative and of fibril solution was positive, which are summarized in In our previous work , the intg and Au ,32,33,34Thereafter, to testify our hypothetical surmise that size or shape may play an important role in such a non-interactive scenario, the dependence of the fibrillary assembly on the sizes of negatively charged spherical iron oxide MNPs, 5 nm, 10 nm and 20 nm was investigated systematically. A fixed amount of LAF of 300 Contrary to the adsorptive results of 5 nm and 20 nm NPs, only the clusters of 10 nm NPs can be found in + and H+ in astrocytes can lead to acidification of endosome, which results in defective amyloid beta (AAlso notice in 3O4 NPs, adsorption and disintegration, upon LAF, and such effects are highly dependent on particle sizes and volume concentration. In essence, the 30 Research presented in this paper demonstrates dual effects of negatively charged carboxyl-group-attached Fe


To compare superb microvascular imaging (SMI) with contrast-enhanced ultrasonography (CEUS) for evaluating the ablation of benign thyroid nodules. 225 Patients with 256 benign thyroid nodules underwent conventional ultrasound, color Doppler flow imaging (CDFI), CEUS, and SMI before and after laser ablation. They were routinely followed up at 1, 3, 6, and 12 months. The volume and volume reduction rate of the ablated nodules was calculated.P< 0.001). CEUS was used as the criterion to determine whether the nodules were completely ablated. The sensitivity, specificity, and accuracy of SMI for detecting incompletely ablated nodules were 90.2, 98.2, and 100%, respectively. The volume of ablated nodules, as measured on ultrasound, was greater than that on CEUS or SMI (both P< 0.001), while CEUS and SMI were similar. The average volume reduction rate of nodules at 1, 3, 6, and 12 months was 40.25, 54.98, 76.83, and 95.43%, respectively. On SMI, the complete ablated nodules had no microvascular perfusion, while the incompletely ablated nodules had microvascular perfusion at the edge of the nodule. The percentages of the detected incompletely ablated nodules of SMI  and CEUS  were comparable, and both were significantly higher than CDFI ( SMI sensitively detected the capillaries within residual thyroid nodules after laser ablation. The lesion size and detection rate of incompletely ablated nodules via SMI was consistent with that of CEUS. SMI may replace CEUS in certain cases for monitoring the curative effect of laser ablation for benign thyroid nodules. Thyroid nodules are common clinical diseases, most of which are benign, but some benign nodules require treatment because of compression symptoms, affecting aesthetics or anxiety of malignant transformation. Although surgical resection is the most common method for treating thyroid nodules, patients are often not willing to accept because of invasion and scarring of the neck. Therefore, nonsurgical and minimally invasive treatment modalities, such as ethanol ablation, percutaneous laser ablation (PLA), and radiofrequency ablation (RFA), have been used to treat thyroid nodules and yielding good results. In 2000, Pacella et al.  were theHowever, determining whether the ablated thyroid nodule is completely necrotized is a problem, as both conventional and color Doppler ultrasound are limited for this purpose. To determine the curative effect of PLA, the microcirculation before and after ablation may be compared via contrast-enhanced ultrasonography (CEUS), reflected as changes in perfusion of the contrast agent. If the lesion is completely ablated, there is no contrast agent perfusion. The efficacy of CEUS for evaluating the completeness of ablation is similar to that of contrast-enhanced computed tomography (CECT) and magnetic resonance imaging (MRI) , 7. HoweA new modality for evaluating microvascular perfusion is superb microvascular imaging (SMI), a noninvasive ultrasonic technology. SMI has great sensitivity for detecting capillaries, which allows better differentiation between slow blood-velocity flow signals and movement artifacts. SMI has been applied for microvascular detection of thyroid, breast, and liver lesions –13 and rCompared with conventional Doppler, SMI shows more accurately the microvascular distribution of thyroid nodules, and its sensitivity for microvascular detection of tissue is superior to that of CEUS . Yet, thThe Medical Ethics Committee of our hospital approved this retrospective study. The requirement for informed consent was waived.This study retrospectively analyzed pathologically proven benign thyroid nodules treated with PLA under the guidance of ultrasound from June 2013 to December 2015 at Third Xiangya Hospital of Central South University.For inclusion in this analysis, patients conformed to the following criteria: thyroid nodule with at least one diameter > 20 mm; neck compression symptoms, including neck discomfort and dysphagia; being with aesthetic requirements but rejected, or were unsuitable, for surgical resection; benign cytological findings (repeated at least twice); and normal thyroid function. Normal thyroid function was considered values within normal range of serum thyroid stimulating hormone (TSH), T3, free triiodothyronine 3 (FT3), T4, and free triiodothyronine 4 (FT4). Patients with any of the following were excluded from this study: coagulation disorders; pathological examination results that indicated malignant nodules; ultrasound findings which showed suspicious malignant nodules, e.g., microcalcification, irregular margins, or anteroposterior-to-transverse diameter ratio (A/T) > 1.The study population comprised 225 patients  with 256 benign thyroid nodules, in which 194 patients had a single nodule and 31 patients had multiple nodules. Multiple nodules in a patient were all located at the same thyroid lobe and were all ablated during the same procedure. μm.After consultation regarding the PLA method, process, postoperative considerations, and the possibility of complications, all the patients provided preoperative informed consent for the PLA operation. PLA was performed with an Esaote EchoLaser integrated ultrasonic diagnosis and laser ablation system . The machine was a MyLab Twice , with an LA332 high-frequency linear array probe, and a frequency of 3.0-11.0 MHz. The laser therapeutic apparatus was an EchoLaser X4 laser treatment system . The Nd:YAG  laser wavelength was 1064 ± 10 nm, the maximum output power of each fiber was 7 W ± 20%, and the length of the fiber was 15 cm with a diameter of 300 With the patient supine, the neck fully exposed, and after sterilizing and anaesthetizing the local skin, a 21G needle  was used to puncture to the center of the targeted nodule under the guidance of ultrasound. The optical fiber was identically positioned through the core needle, and the guided needle stepped back 5 mm so that the tip of the optical fiber contacted the tissue directly.After setting the relevant parameters of the laser light source  and opening the laser emission, the treatment was begun. The number of ablation fibers and total energy was determined based on the shape and size of the nodules. In most patients, a nodule <15 mL could be ablated by a single fiber with the pinpoint located in the geometric center of the nodule. A nodule ≥15 mL was generally ablated by more than 2 fibers simultaneously. When multiple fibers were needed, they were parallel and within 10 mm of each other, depending on the shape of the nodule. If the nodule was large, all pinpoints and optical fibers could be synchronously drawn back 10 mm to conduct a second ablation based on the original needle arrangement, when necessary.The entire treatment procedure was monitored by real-time ultrasound. As the energy is released from the laser light source, an irregular hyperechoic gasification area forms at the tip of the optical fiber, which expands. The treatment was ended when this area completely covered and was larger than the targeted nodule.In this study, CDFI and SMI were first performed for the patients one hour after ablation, followed by CEUS, and the curative effect of ablation was evaluated. As the postoperative pathological results were unavailable in this study, whether the nodule was completely ablated was determined based on CEUS, as follows. A completely ablated nodule was defined as a nodule without enhancement and with no contrast agent filling, and its range was larger than the original size of the nodule. An incompletely ablated nodule was identified as a small amount of residual tissue with irregular contrast agent perfusion along the margins of the nodule.1) was calculated based on the following formula: V1 = π·ghi / 6, where g, h, and i were the length, width, and height, respectively.Prior to CEUS, SMI was performed by a sonographer with >5 years' experience. A Toshiba Aplio 500 ultrasonic diagnostic instrument  was used, with a 14L5 linear array probe and a frequency 14 MHz. SMI included both color and monochrome SMI, and the monochrome  mode has improved sensitivity by subtracting the background information and focuses only on the vasculature. The probe was placed above the lesion and the sampling range was adjusted to cover the entire lesion and surrounding thyroid parenchyma. The color-gain was broadened as far as possible to avoid noise. Microvascular distribution within the ablated lesion was observed using a low-speed scale (1.0-2.0 cm/s) and it was thus determined whether there was residual tissue. The size of the area without blood flow on SMI was measured, and the volume of this region , with a 9L4 linear array probe and a frequency of 9 MHz. The size of the ablated nodule was measured on 2D ultrasonic images. The volume (V) of the target nodule was calculated as: V = The dynamic double contrast-imaging model was adopted with contrast-tuned imaging. The mechanical index was 0.03. The contrast agent was the second generation of SonoVue , i.e., sulfur hexafluoride microbubbles enclosed by phospholipids. Twenty-five milligrams of SonoVue was diluted with 5 mL saline (0.9% Na Cl), and after vibrating for 30 s, a microbubble suspension was created. The suspension was injected as a bolus through the antecubital vein with a 20 G trocar; 2.4 mL for each injection was then flushed with 5 mL saline. The dynamic perfusion of the lesion was continuously observed for 90 s, and the images were stored.2) was calculated as V2 = π·def/6, where d, e, and f were the length, width, and height of the area, respectively.Whether there was residual tissue was determined by the enhanced signal within the ablated lesion. The size of the nonenhanced area on CEUS was measured, and the volume  was calculated as VRR = [(preoperative volume – volume at follow-up) / preoperative volume] × 100%. The thyroid function was tested before and one month after ablation. t-test. The rates of detected incomplete ablated nodules among the CDFI, CEUS, and SMI images were compared using McNemar's test. Calculations were performed for the sensitivity, specificity, and accuracy of SMI for diagnosing the accuracy of detecting incomplete ablation nodules. One-way analysis of variance was used to compare changes in nodule volume and VRR. Statistical significance was defined as P < 0.05.All statistical analyses were performed using SPSS for Windows software . All values are shown as mean ± standard deviation. Comparisons of the volume of the ablated nodule among the CDFI, CEUS, and SMI images were performed using the pairedIn this analysis, 225 patients (256 benign thyroid nodules) conformed to the study criteria . All theOn CDFI, there were color blood flow signals within all nodules before ablation. The color Doppler signals within completely ablated nodules were absent after PLA, while blood flow signals were still present at the margins of some incompletely ablated nodules Figures  and 2.On CEUS, prior to PLA there were contrast agent perfusions within all nodules. After PLA, there were no contrast agent fillings and no enhancements within completely ablated nodules, and the regions of nodules were larger than the original sizes Figures  and 2. TOn SMI, before PLA, there were microvascular perfusions within all nodules. After PLA, there were no microvascular perfusions within completely ablated nodules, and the regions of nodules were larger than the original sizes. In incompletely ablated nodules there were residual tissues with microvascular perfusions Figures  and 2.P = 0.481), but both rates were significantly higher than that of CDFI (P< 0.001). In this study, CEUS was used as the criterion to determine whether the nodules were completely ablated. The sensitivity, specificity, and accuracy of SMI for detecting incomplete ablation nodules were 90.2%, 98.2%, and 100%, respectively. Supplementary ablations were performed for 41 incompletely ablated nodules 24 hours after the first ablations.Of the 256 nodules, 10, 37, and 41 were detected as incompletely ablated on CDFI, SMI, and CEUS, respectively . The difference in detection rates between the SMI and CEUS was not significant ( P< 0.001). The mean volume of ablated nodules on CEUS and SMI was comparable (P = 0.533).The postoperative volumes of ablated nodules were 15.10 ± 5.25mL on 2D-ultrasound, 12.15 ± 3.83 mL on CEUS, and 12.98 ± 4.34 mL on SMI. The mean volume of ablated nodules as measured on 2D-ultrasound was significantly greater than that of CEUS or SMI .As measured on 2D-ultrasound, the postoperative volume of nodules differed significantly from that of the preoperative volumes (allIn this study, the completeness of ablation of benign thyroid nodules was evaluated and compared between SMI and CEUS. On SMI, microvascular perfusion was absent within completely ablated nodules, but present in incompletely ablated nodules at the margins of residual tissues. The rates of detection of incompletely ablated nodules using SMI and CEUS were comparable, and both of these were significantly higher than that of CDFI. When adopting CEUS as the criterion to determine whether the nodules were completely ablated, the accuracy, sensitivity, and specificity of using SMI for evaluating complete ablation was 100%, 90.2%, and 98.2%, respectively.CDFI is not accurate for depicting the blood flow within the nodule, as there may be significant artifacts due to the heating effects and tissue gasification immediately after the ablation. In addition, CDFI employs a wall filter to eliminate noise and motion artifacts, which can easily cause loss of sensitivity for showing low-velocity blood flow in microvessels .The ablation is ended when the transient hyperechoic area, produced by gasification of the tissue and cells exposed to heat, completely covers and exceeds the nodules , 18. AftAlthough CEUS is a preferred method for evaluating the effect of ablation, it is invasive and requires administration of contrast agent. Compared with traditional Doppler ultrasound, SMI technology can more accurately display greater details of microvascular flow and vessel branching in the peripheral and internal microvasculature of thyroid nodules, by identifying noises associated with blood flow and tissue movement and using an adaptive calculation method . SMI simThe new SMI technology has relatively improved signal-to-noise ratio, and may be able to detect microvascular blood flow signals ≥ 0.1 mm/s . Machadoμm and blood flow of ~1 mm/s [In the present study, SMI failed to detect four incompletely ablated nodules that were shown on CEUS. Studies have shown that CEUS can show capillaries with a diameter < 40  ~1 mm/s . The senThe present study found that the volume of ablated lesions revealed by 2D-ultrasound was greater than the volume shown on CEUS or SMI. This is consistent with the conclusion of an experimental study with rabbit liver . The reaThis study is limited in several ways. First, we did not have available postoperative pathology results, which are the gold standard for determining the presence of residual nodules. Second, the contrast-enhanced and super microvascular images were performed with different ultrasound devices, which may have affected our results. Finally, we only evaluated the short-term efficacy of laser ablation, and a long-term follow-up is important for determining the presence of residual nodules.In conclusion, SMI can sensitively detect the capillaries within residual thyroid nodules after laser ablation, and the rate of detection for incompletely ablated nodules and evaluation of lesion size was consistent with that of CEUS.


Oryza sativa var. indica). We found that N delays germination from 0 to 12 h in a genotype-dependent and source-dependent manner, especially with urea and nitrate. We identified contrasting groups of fast germinating genotypes such as Aditya, Nidhi, and Swarnadhan, which were also least delayed by N and slow germinating genotypes such as Panvel 1, Triguna, and Vikramarya, which were also most delayed by N. Oxygen uptake measurements in the seeds of contrasting genotypes revealed that they were affected by N source in accordance with germination rates, especially with urea. Germinating seeds were found to have endogenous urease activity, indicating the need to explore genotypic differences in the effective urea uptake and metabolism, which remain unexplored so far. Urea was found to significantly inhibit early root growth in all genotypes but not shoot growth. Field evaluation of 15 of the above genotypes clearly showed that germination rates, crop duration, and yield are linked to NUE. Slow germinating genotypes had longer crop duration and higher yield even at lower N, indicating their higher NUE, relative to fast germinating or short duration genotypes. Moreover, longer duration genotypes suffered lesser yield losses at reduced N levels as compared to short duration genotypes, which is also a measure of their NUE. Together, these results indicate the potential of germination rates, crop duration, urea utilization and its effect on root growth in the development of novel phenotypic traits for screening genotypes and crop improvement for NUE, at least in rice.The biological improvement of fertilizer nitrogen use efficiency (NUE) is hampered by the poor characterization of the phenotype and genotype for crop N response and NUE. In an attempt to identify phenotypic traits for N-response and NUE in the earliest stages of plant growth, we analyzed the N-responsive germination, respiration, urease activities, and root/shoot growth of 21 Indica genotypes of rice ( While t cereals , consumiiculture . The ideiculture . There hiculture , dense aiculture  etc.vis-à-vis yield; the mechanism involves oxygen consumption driven acceleration of the meristematic growth (starting with germination), which in turn hastens the life cycle such as branching, such that the plant matures faster and yield is lowered due to shortened life span of the photosynthetic plant  and yields (3.6–7.8 t/ha). Of these 21, the seeds of 20 genotypes were procured from the Indian Institute of Rice Research, Hyderabad, India. They were, Aditya, Swarnadhan, Nidhi, Jaya, Vikas, Ajaya, Krishnahamsa, Mandyavijaya, Nagarjuna, Prasanna, Pusa Basmati, Ravi, Rasi, Sampada, Suraksha, Swarna, Triguna, Varadhan, Vibhava, and Vikramarya. Seeds of the genotype Panvel 1 were procured from Kharland rice research station, Panvel, Maharashtra, India. All the 21 genotypes were used for germination studies in the lab, following which contrasting pairs were identified and used for oxygen consumption, growth, and urease activity measurements. Independently, 15 of these genotypes were evaluated in the field at the Indian Institute of Rice Research, Hyderabad, as detailed under “Field experimental conditions” (see below).Twenty-one rice genotypes of 3 (5 mM) and Ca (NO3)2 (5 mM), which were replaced with urea (7.5 mM), or NH4NO3 (7.5 mM), or NH4Cl (15 mM) as the sole N source, at an equalized concentration of 15 mM. Distilled water and N-free media were used as controls. The Petri plates were incubated in a plant growth chamber at 28°C, 75% humidity, 600 lux light intensity at the plant level, obtained from Osram fluorescent tubes and 12 h/12 h photoperiod.Seeds of the selected rice genotypes were weighed individually and only seeds of modal weight were used as described earlier  for all 3 5.01%; CEC 44.1 C mol (p+)/kg soil; soil organic carbon 0.70%; Soil available N 215 kg/ha; available phosphorus 46 kg P/ha; potassium 442 kg K/ha, and zinc 12.5 ppm.Fifteen of the above mentioned Indica rice genotypes were used for field evaluation, namely Aditya, Swarnadhan, Rasi, Jaya, Varadhan, Ravi, Swarna, Suraksha, Vibhava, Vikas, Krishna Hamsa, Sampada, Prasanna, Pusa Basmati, and Triguna. This evaluation was conducted at the farm of the Indian Institute of Rice Research, Hyderabad, India, for six seasons over 3 years  from 2010–2011 to 2014–2015. The geographical coordinates of the experimental farm are, 17°19″ N latitude and 78°23″ E longitude, at an altitude of 542 m above sea level and mean annual precipitation of 750 mm. The characteristics of field soil was pH 8.1; EC 0.7l dS/m; free CaCOThe field experiment was based on a split plot design of plot size 7.0 m × 3.5 m with three replicates. Urea was the sole source of N, applied at the rate of 100 kg N/ha (N100) or 217 kg urea/ha in three equal splits , with a control of no added N (N0). To maintain isolated conditions between plots, the field was divided into two separate blocks of N-0 and N-100 by making a deep trench of 4 feet between them and placing thick polythene sheets in the trench deep into the soil to avoid leaching from plot to plot and these plots were being maintained permanently. Moreover, the same exact plots were used for N0 and N100 throughout the six seasons. Yield and crop duration was measured using a sample of one meter square from two different areas of each plot, containing approximately 66 plants per sample.½). It was calculated by interpolating the data using the X0 function in SigmaPlot software version 9. Oxygen consumption was measured as the rate of oxygen consumption at the t½ time point for selected contrasting rice genotypes using Oxygraph 2.1 from Hansatech, United Kingdom. For this purpose, four seeds each of modal weight were taken in duplicates in cuvettes filled with media with or without nitrate/urea, and O2 consumption was monitored for 12 min for every measurement.The number of seeds germinated in each plate of 100 seeds in triplicate (300 seeds per treatment) was monitored every 3 h in terms of the visible emergence of the radical, until all the seeds in the plate visibly germinated. The germination rate is defined as the time taken for 50% seeds to germinate .Qualitative urease assay was performed by phenol red indicator method using germinating seeds of the genotype Panvel 1. For this purpose, triplicate sets of 100 seeds each were soaked in autoclaved deionized water for 2 h and plated on Arnon Hoagland media with or without urea as the sole N source. At 72 h, when two-thirds of them showed visible signs of germination, they were qualitatively tested for urease using either intact or crushed seeds in an assay mixture that contained urea broth and Arnon Hoagland media in equal proportions (with or without urea as the sole N source). The media in which the seeds were grown were also tested to ensure that there was no urease activity due to microbial contamination during the experiment. As a positive control, garden soil sample was tested and change of color was observed. Quantitative estimation of endogenous urease activity was done by spectrophotometric detection of ammonium ions released, as described by ½ or the time taken for 50% seeds to germinate . In other words, longer duration genotypes suffer lesser yield loss under N0 relative to N100, indicating higher NUE at longer crop duration. This observation is significant at P < 0.05 .Field evaluation of 15 of these above 21 genotypes for crop duration and yield (½), the extent of N-induced delay in 50% seeds to germinate  and crop duration.It must be emphasized that while the lab experiments on germination used totally N-depleted or N-replete media, the N0 condition in field experiments only indicates that there was no added N. The residual N in the soil depletes only gradually over 3 years of field experiments and therefore, N0 condition denotes low-N rather than zero-N. Nevertheless, considering that NUE is best assessed under low-N conditions, the finding that some genotypes gave higher yield under N0 than others, or suffered lesser yield loss under N0 than others, is significant from the NUE standpoint. We found that the longer duration genotypes Vardhan, Pusa Basmati, Krishnahamsa, and Triguna gave significantly higher yield in the field under N0 condition, indicating higher NUE, as compared to the short duration genotypes Aditya, Swarnadhan, and Rasi, whose germination was least delayed by N. More importantly, we identified at least 3 novel phenotypic traits associated with such NUE: time taken for 50% seeds to germinate (t2 uptake as compared to slow germinating (long duration) genotypes , while N input inhibited O2 uptake. Urea inhibited O2 uptake the most, while it was only moderately inhibited by nitrate, as compared to media without N . These effects were more significant with urea rather than with nitrate, and the degree of inhibition between fast and slow genotypes did not reveal any contrasting pattern (Table 4). Nevertheless, it is clear that the observed differences in the germination rates between contrasting genotypes can be accounted largely by their oxygen consumption and additionally by the influence of N on oxygen consumption, germination, or both.Germination is driven primarily by hydration and energy utilization and is a oxygen consumption-intensive phase in the development of a plant . The rolFigure 7A). Even though urease activity was found in the extracts of crushed seeds , its activity was orders of magnitude lower than in soil and presumably inadequate to produce any metabolic effect through a downstream product of urea metabolism. Whether this chemical effect of urea was direct or through a signaling cascade is an interesting aspect for further study.As the seeds were germinated in Petri plates on cotton moistened with sterilized media, the inhibitory effect of N in the form of urea is expected to be a chemical effect of urea uptake rather than a metabolic effect, unlike farm soil, in which microbial urease activity plays a major role. This was confirmed by the lack of detectable urease activity in the external media, indicating that the observed urea effect was the result of uptake of urea . Secondly, the urea effect is seen even in the absence of external urease in our case , while the soil urease may be very high in their case. Thirdly, the very low level of urease in more than 2 g crushed seeds as compared to the high activity even in a few milligram of soil (control) shows that endogenous urease was inadequate to produce ammonia toxicity in our case .In Arabidopsis seeds, P < 0.05, student’s t-test, Figure 7C), despite internal variation between individual genotypes. The presence of urea transporters  genotypes had significantly lower urease activity (by 18.5%) as compared to the fast germinating (short duration) genotypes . While it revealed significant effects of N on both shoot and root length when all the N treatments and genotypes were taken together , there was only significant inhibition of root length but not shoot length  by any individual N source on any or all individual genotypes . Urea not only had the most inhibitory effect on root length , but also showed a dose–response relationship , which was not found with shoot length . Moreover, there was differential inhibition of root length by urea in contrasting groups of genotypes despite individual variations, indicating the need for further exploration of its potential for screening. The molecular mechanisms of these findings are also worthy of further exploration, since inhibition of cyanide insensitive oxygen consumption by alternative oxidase in rice was also shown to inhibit root growth , the influence of N on it (Δt½) and crop duration determine yield and NUE, at least in the Indica subspecies of rice. They can be further evaluated on a larger scale for their use as simple phenotyping tools to screen the rice germplasm for NUE. The fact that the 21 Indica genotypes used in this study represent diverse agroclimates, crop durations, and yields lends further credence to such a conclusion. The effect of N on O2 consumption points to the regulation of oxygen consumption as a possible mechanism for further investigation. The genotypic differences in urea uptake/utilization and urease activities remain a novel observation for further research in a hitherto unexplored area of research.Our findings provide adequate evidence that the germination rate (tNS performed most of the experiments, data analysis, and wrote the first draft. VBS helped in early germination experiments. NG performed biplot analysis. SR performed qualitative urease tests. SK conducted the field experiments. VS suggested the exploration of the role of germination and respiration in NUE. RP provided statistical suggestions. NR helped in the planning, mentoring and supervision of the experiments, data interpretation and manuscript preparation.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.


Pheochromocytomas (PCC) and paragangliomas (PGL) are rare neuroendocrine tumors that arise in the adrenal medulla and in extra-adrenal locations, such as the head, neck, thorax, abdomen, and pelvis. Classification of these tumors into those with or without metastatic potential on the basis of gross or microscopic features is challenging. Recent insights and scoring systems have attempted to develop solutions for this, as described in the latest World Health Organization (WHO) edition on endocrine tumor pathology. PCC and PGL are amongst the tumors most frequently accompanied by germline mutations. More than 20 genes are responsible for a hereditary background in up to 40% of these tumors; somatic mutations in the same and several additional genes form the basis for another 30%. However, this does not allow for a complete understanding of the pathogenesis or targeted treatment of PCC and PGL, for which surgery is the primary treatment and for which metastasis is associated with poor outcome. This review describes recent insights into the cell of origin of these tumors, the latest developments with regard to the genetic background, and the current status of tumor classification including proposed scoring systems.  SDHx) mutations.Pheochromocytomas (PCC) are rare neuroendocrine tumors that, by definition, arise in the adrenal medulla; however, the term paraganglioma (PGL) is reserved for tumors that arise from sympathetic ganglia in the thorax, abdomen, and pelvis as well as from parasympathetic paraganglia in the head and neck area. Although these tumors are rare, with an incidence of approximately five per million, they are relevant from various perspectives, including pathophysiology and genetic background. Such tumors also have a relationship with various other tumors, such as neuroblastomas, that also originate from the adrenal medulla and extra-adrenal sympathetic tissues as well as tumors co-occurring in various hereditary tumor syndromes, for instance, medullary thyroid carcinoma in MEN2, renal cell carcinoma in VHL disease, and gastrointestinal stromal tumors (GIST) in the context of succinate dehydrogenase subunit gene , somatic (s), EPAS1/HIF2 alpha  and EGLN1/2 (g). The TCA-cycle-related subgroup includes mutations in genes encoding energy metabolism enzymes such as SDHx (g), FH (g), MDH2 (g) and IDH1/2 (s). Inactivating mutations lead to an accumulation of TCA metabolites causing epigenetic changes in HIF stability and are associated with a hypermethylated phenotype [This cluster represents germline or somatic gene mutations resulting in a dysfunctional hypoxic (pseudo-hypoxic) response with a central role for hypoxia inducible factor (HIF)1 alpha and HIF2 alpha, which are the main components of the response to low oxygen levels. Under normal conditions, HIF1 alpha and HIF2 alpha are in an inactivated state. Under hypoxic conditions or in the context of a dysfunctional system, HIFs are stabilized and activate target genes inducing angiogenesis, metabolism, apoptosis, and proliferation. The cluster can be divided into two subgroups. The henotype ,24,25,27VHL gene encodes two proteins, pVHL30 and pVHL19. pVHL plays a critical role in degradation of HIF alpha, and pVHL dysfunction results in a pseudohypoxic response. The VHL gene is known to cause von Hippel-Lindau (VHL) disease, an autosomal dominant familial tumor syndrome characterized by multiple benign and malignant tumors including retinal and central nervous system hemangioblastomas, renal cysts, clear cell renal cell carcinomas (RCC), pancreatic cysts, pancreatic neuroendocrine tumors, endolymphatic sac tumors, and epididymal cystadenomas. VHL disease shows a genotype–phenotype correlation; VHL type 2 is characterized by missense mutations and is linked to PCC, which is observed in 10–26% of VHL patients. Germline mutations in VHL account for 5–10% of hereditary PCC/PGL, and somatic VHL mutations have been found in about 10% of sporadic PCC/PGL [The  PCC/PGL ,29.EPAS1/HIF2 alpha gene encodes for HIF2 alpha and mutations lead to its reduced degradation and stabilization. Somatic EPAS1/HIF2 alpha mutations have been found in 5–10% of PCC/PGL. In addition, germline mutations have been identified, but they comprise <1% of PCC/PGL. EPAS1/HIF2 alpha mutations are also linked to polycythemia and somatostinoma [The ostinoma ,31,32. EGLN1 and EGLN2 genes encode for PHD2 and PHD1, respectively. PHD proteins are involved in the degradation of HIFs by hydroxylation. Germline mutations in EGLN1 and EGLN2 have been identified in <1% of PCC/PGL and are also associated with polycythemia [The cythemia ,34. SDHx family are currently the most common germline mutations found in hereditary PCC/PGL and account for about 20–30% of hereditary cases. Somatic mutations have rarely been reported [SDHx-related PCC/PGL are caused by mutations in five genes of the SDHx family, SDHA, SDHB, SDHC, SDHD, and SDHAF2, which encode the corresponding proteins. The SDH complex, composed of the four subunits SDHA–D, is a key component of the TCA cycle and the electron transport chain and is located at the inner mitochondrial membrane. SDHAF2 is essential in the assembly of the complex. The complex catalyzes the oxidation of succinate to fumarate. Loss of SDH function results in accumulation of succinate, which inhibits the prolyl hydroxylation of HIF1 alpha, resulting in a hypoxic response [SDHx family are predominantly linked to extra-adrenal PGLs. SDHB mutations are associated with abdominal and thoracic PGL and show a 30–70% risk of metastasis. SDHD and SDHA mutations are mostly found in abdominal, thoracic, head, and neck PGLs. SDHC and SDHAF2 mutations are associated with head and neck PGLs [SDHD and SDHAF2 genes, both located on chromosome 11, show a parent-of-origin-dependent inheritance effect in which mutations almost exclusively cause disease after paternal transmission. Somatic loss of maternal chromosome 11 plays an important role in this effect according to the Hensen model [SDHx family have also been associated with other tumors such as SDH-deficient GIST, SDH-deficient RCC, and SDH-deficient pituitary adenoma [Mutations in the reported . SDHx-reresponse ,37. Mutaeck PGLs . Mutatioen model ,39. Muta adenoma . SDHA germline mutations [SDHC promoter methylation in the absence of SDHx germline mutations. In addition, SDHB negative PCC/PGL without known pathogenic SDHx mutations or SDHC promoter methylation have been described, suggesting that SDHB IHC might be of additional value in the assessment of SDHx genetic variants of unknown significance [If the SDH complex becomes instable by dysfunction of any of the components, the SDHB subunit is degraded in the cytoplasm. The loss of SDHB expression can be detected by SDHB protein immunohistochemistry (IHC); this can be used to detect tumors with mutations in one of the five genes. In addition, loss of SDHA IHC can detect tumors with utations ,41,42,43FH gene encodes fumarate hydratase, a TCA enzyme which converts fumarate into malate. Mutant FH results in accumulation of fumarate leading to PHD inactivation and HIF stabilization. Mutations in this gene have classically been associated with hereditary cutaneous and uterine leiomyomatosis and a specific variant of RCC, which is regarded as a separate entity by the WHO [FH-mutated PCC/PGL [FH mutations, suggestive of altered DNA methylation and protein succination, respectively [MDH2 gene encodes MDH2, another TCA enzyme that catalyzes the reversible oxidation of malate to oxaloacetate. MDH2 dysfunction leads to accumulation of metabolites and a hypermethylator phenotype. Germline mutations have been identified in less than 1% PCC/PGL and show undetectable 5-hmC staining. The isodehydrogenase (IDH) enzymes IDH1 and IDH2, encoded by IDH1 and IDH2, convert isocitrate to alpha-ketoglutarate in the TCA cycle. Mutations result in the production of 2-hydroxyglutarate instead of alpha-ketoglutarate, and its accumulation activates the hypoxic pathway. Somatic IDH1 and IDH2 mutations are typically present in low-grade and secondary high-grade gliomas and have rarely been found in PGLs [The  the WHO . Recentl the WHO . FH IHC  PCC/PGL . In addiectively . The MDH in PGLs ,49. SLC25A11 gene encodes the mitochondrial 2-oxoglutarate/malate carrier and germline mutations have been identified in abdominal as well as head and neck PGLs and may be associated with metastases [IDH3B gene encodes IDHB3, an enzyme involved in the oxidation of isocitrate to alpha-ketoglutarate in the TCA cycle, and a germline mutation was identified in a jugular PGL [GOT2 gene has been found in metastatic abdominal and thoracic PGL with a high succinate/fumarate ratio in tumor cells [DNMT3A gene plays a role in DNA methylation during embryonic development and germline mutations have been found in multifocal PGLs [DLST gene encodes a mitochondrial protein that belongs to the 2-oxoacid dehydrogenase family. Germline mutations have recently been found in PCC/PGL with an expression and methylation profile suggestive of the pseudohypoxic pathway. In addition, positive DLST immunostaining was detected in tumors with TCA-cycle or EPAS1 mutations [Germline mutations in other genes with a TCA-related function and/or a hypermethylated phenotype have been recently described in 1% or fewer of PCC/PGL and might be included in this cluster. The tastases . The IDHular PGL . GOT2 isor cells . The DNMcal PGLs . The DLSutations .RET (g/s), NF1 (g/s), MAX (g/s), MEN1 (g), TMEM127 (g), H-RAS (s), and KIF1B (g/s). These mutations are associated with the activation of the RAS-RAF-ERK, PI3K-AKT-mTOR , and MYC-MAX (MAX) kinase signaling pathways. The activation or deregulation of these pathways leads to uncontrolled proliferation, growth, and cell survival [The common denominator for Cluster 2 is the activation of kinase signaling pathways. It encompasses germline or somatic mutations in survival ,23,24,25RET proto-oncogene encodes a transmembrane tyrosine kinase. When ligand binds to the RET receptor or if there is an activating mutation, a signaling cascade is triggered through the PI3 kinase pathway to regulate cell proliferation and apoptosis. Activating germline mutations in the RET gene cause the multiple endocrine neoplasia type 2 (MEN2) syndrome characterized by the development of PCC and medullary thyroid carcinoma. PCC occur in 40–50% of patients with MEN2 and the risk of PCC is associated with specific RET mutations. Clues to MEN2-related PCC include the presence of multiple, bilateral tumor nodules and adrenal medullary hyperplasia. Germline RET mutations have been detected in 5% of hereditary PCC/PGL and somatic mutations in about 5% of sporadic tumors [The c tumors ,53,54.NF1 gene encodes neurofibromin 1, which is a negative regulator of the RAS intracellular signaling pathway. Inactivating mutations in the NF1 gene disrupt this inhibitory effect. NF1 is the most common somatically mutated gene in PCC/PGL, accounting for 20–40% of sporadic PCC/PGL. However, only approximately 6% of patients with the autosomal dominant NF1 syndrome, characterized by germline NF1 mutations, develop PCC, and germline mutations account for <5% of all PCC/PGL. Interestingly, up to 16% of NF1-related PCC show a composite histological phenotype of typical PCC intermixed with areas of ganglioneuroblastoma or ganglioneuroma [The oneuroma ,55. TMEM 127 is a tumor suppressor gene linked with the mTOR kinase pathway, and germline mutations have been found in less than 5% of PCC/PGL. It encodes a transmembrane protein that functions as a negative regulator of mTOR. Mutant forms of the protein are nonfunctional and cause cytoplasmic localization of the protein. Notably, mutant alleles show relatively low penetrance [netrance ,57.MAX is a tumor suppressor gene with a regulatory role in the MYC-MAX-MD1 network. Truncated MAX mutations can cause dysregulation of the mTOR pathway. Germline mutations, which may exhibit a parent-of-origin effect with paternal transmission, as well as somatic mutations have been found in PCC/PGL, both in less than 5% of cases [MAX mutations in patients in case of absent immunostaining, comparable to negative SDHB staining, this has not found widespread application [of cases ,59. AlthH-RAS is a proto-oncogene that encodes H-RAS protein, which can bind to GTP and activate the RAS/RAF/ERK signaling pathway leading to cell proliferation. Missense somatic mutations have been found in 5–10% of sporadic PCC/PGL [ PCC/PGL ,61.KIF1B gene encodes for two proteins isoforms, KIF1B alpha and KIF1B beta. KIF1B alpha is involved in the transport of mitochondria, and KIF1B beta in the transport of synaptic vesicle precursors. In addition to its transport function, KIF1B beta plays a role in apoptosis and dysfunctional KIF1B beta due to mutations in the KIF1B gene and can lead to tumorigenesis. The KIF1B gene might be one of the most frequently somatically mutated genes in PCC/PGL, with somatic mutations seen in up to 20% of cases, and also germline mutations have been described in fewer than 1%. KIF1B mutant PCC/PGL shows a similar transcription profile as RET and NF1 mutant PCC/PGL [The  PCC/PGL ,63. MEN1 gene codes for the menin protein, which interacts with transcription factor JunD. JunD is a functional component of the AP1 transcription factor complex. It has been proposed to protect cells from p53-dependent senescence and apoptosis. Germline mutations in the MEN1 gene lead to MEN1 syndrome, characterized by various types of endocrine and non-endocrine tumors, including pituitary adenomas, hyperplastic parathyroids, and neuroendocrine pancreatic tumors, with a high penetrance of disease but PCC/PGL are rare and account for <1% of PCC/PGL [The  PCC/PGL ,65,66. All PCC/PGL are believed to exhibit some metastatic potential, but only a subset of these tumors will actually metastasize. Until 2004, malignancy was defined by either metastasis or on the basis of extensive local invasion. The 2004 WHO classification defined malignant PCC by the development of metastasis. Due to incongruity, confusion was noted and the current WHO classification encourages the use of the terms “metastatic” and “non-metastatic”. Many histomorphological features and immunohistochemical markers have been studied to find biomarkers to differentiate between metastasizing and non-metastasizing PCC/PGL. However, no single histomorphological or immunohistochemical feature has indicated metastatic potential. Therefore, several algorithms, on the basis of multiple histologic properties, have been described to detect potential for aggressive behavior. In 1990, Linnoila examined 120 PCC/PGL and developed a statistical model to predict malignancy. According to this model, there was a 95% probability that more than 70% of tumors could be classified correctly on the basis of four factors: extra-adrenal location, coarse nodularity, confluent tumor necrosis, and absence of hyaline globules. The majority of malignant PCC/PGL had two or three of these features (71%) while 89% of benign tumors had none or one . UnfortuThompson described the Pheochromocytoma of the Adrenal gland Scaled Score (PASS), which consists of twelve parameters: large nests or diffuse growth, central or confluent necrosis, high cellularity, cellular monotony, tumor cell spindling, >3 mitotic figures/10 high power fields, atypical mitotic figures, extension into adipose tissue, vascular invasion, capsular invasion, profound nuclear pleomorphism, and nuclear hyperchromasia. A maximum score of 20 points is obtained when all features are present. Tumors with a score <4 were considered to have no metastatic potential. Tumors with a score ≥4 were considered to have an increased metastatic potential, as seen in Commonly agreed upon features are the presence of atypical mitosis, necrosis, capsular involvement, lymphovascular invasion, and extension into adipose tissue. However, significant interobserver and intraobserver variability is described in scoring the presence or absence of high cellularity, nuclear pleomorphism, and hyperchromasia . A meta-Although PASS only applies to PCC, the Grading of Adrenal Pheochromocytoma and Paraganglioma (GAPP) is a tool for PCC as well as PGL. It is based on histological pattern, cellularity, comedo-type necrosis, capsular invasion, vascular invasion, Ki67 labelling index and catecholamine type. On the basis of a maximum score of 10 points, tumors are graded as well differentiated (0–2 points), moderately differentiated (3–6 points), and poorly differentiated (7–10 points), as seen in A sensitivity of 50% and specificity of 80% has been described for PCC with GAPP. In addition, the PPV was 5% and NPV was 99%. For PGL, the sensitivity was 100% and the specificity was 68%, with a PPV of 29% and a NPV of 100% . Both PASS and GAPP do not account for the effect of underlying somatic or germline mutations, whether these are “low risk driver mutations” or “high risk driver mutations”. For example, PCC in MEN 2A typically have a low risk of metastases, with an estimated prevalence of 3% . HoweverSDHB mutations; the presence of SDHB mutations should be considered a risk factor for metastases or recurrence [SDHx mutations. Loss of SDHB staining is not seen either in prognostically unfavorable SDHB-mutated tumors or in all tumors with SDHx mutations; however, SDHB IHC negativity by itself has also been correlated with metastases [MAML3 fusion gene variants, leading to Wnt signaling upregulation, as seen in Cluster 3, described above, are associated with metastases. In the same study, Ki67 was found to be correlated with metastatic disease. Interestingly, the tumor with the highest Ki67 expression was MAML3 fusion-positive [RASSF1A, NORE1A, p16INK4A, RARB, DCR2, CDH1, and APC. Methylation of three or more promoters was found in five PGL, of which four were metastatic [Recurrence and metastases are strongly associated with currence . As desctastases ,43. In atastases . Additiopositive . Anothertastatic . DespiteSDHB mutations [SDHx mutations, and increased risk has not been noted for SDHA, SDHC, and SDHD mutations. Both PASS and GAPP have a low PPV but a high NPV, suggesting that these models are good at ruling out but poor at predicting metatastatic potential.Recently, PCC and PGL have proven to be enigmatic tumors with regard to the prediction of their clinical behavior; they are also exciting tumors with regard to their pathophysiology and genetic background. Major progress has been made in various fields, although the survival of metastatic PCC and PGL is still bleak. In the field of PCC and PGL genetics, the number of genes involved continues to increase, with a particular focus on enzymes and other proteins that are involved in the TCA cycle. With the advent of whole exome and whole genome sequencing, more such genes may be detected, all of which may potentially converge along the same pathophysiological pathway. Whole exome sequencing is expected to become the standard of genetic analysis in the next 5–10 years, allowing rapid detection of mutations in PCC and PGL patients and families. IHC, for instance, with SDHA and SDHB antibodies, may serve as a confirmatory technique with regard to the classification of variants of unknown significance. With regard to classification, the most important new developments can be found in the 2017 edition of the WHO volume on Endocrine Tumors, in which all PCC and PGL are now considered to have some metastatic potential and that the pathological grading system for PCC and PGL (GAPP), developed by Kimura, has been independently validated. In addition, a modified GAPP (M-GAPP) has been proposed, incorporating loss of SDHB staining as a criterion, which seems logical in light of the increased metastatic risk related to utations . HoweverFinally, although outside the scope of this review, the improved knowledge of the genetic background and the genes involved in the pathogenesis of PCC and PGL will allow for the development of targeted therapies for those tumors that cannot be resected surgically or that have metastasized, potentially improving prognosis for these patients.