Purpose: Pheochromocytomas (PCCs) are neuroendocrine tumors arising from the adrenal medulla or as paraganglioma (PGL) from extra-adrenal sites. While usually benign, a small fraction is malignant. Multi-modality therapy is used in treating malignant disease; however, little data exist on the role of external beam radiation therapy (EBRT). In this retrospective review, we assessed response to EBRT in malignant PCCs or PGLs.Methods and Materials: Records of patients treated at the National Institutes of Health who received EBRT between 1990 and 2012 were studied. Patients were assessed for symptomatic control, biochemical response, local and distant control by response evaluation criteria in solid tumors v1.1 or stable disease on imaging reports, toxicity by radiation therapy oncology group (RTOG) criteria, and survival.Results: There were 24 patients treated who received EBRT to lesions of the abdomen (n = 3), central nervous system (n = 4), and bone (n = 40). Lesions were treated with 3D conformal EBRT to a mean dose of 31.8 Gy in 3.3 Gy fractions, or fractionated stereotactic radiosurgery to 21.9 Gy in 13.6 Gy fractions. Patients experienced acute (n = 15) and late (n = 2) RTOG toxicities; no patient experienced acute toxicity ≥4 or late toxicity ≥2. Symptomatic control was achieved in 81.1% of lesions. Stable radiographic response was achieved in 86.7% of lesions with progression in 13%. Distant progression was observed overall in 75% of patients and average survival was 52.4 months.Conclusion: Malignant PCC and PGL often do not respond well to current systemic therapies. In these cases, EBRT can be considered in patients with symptomatic, localized disease progression. Pheochromocytomas (PCCs) are neuroendocrine tumors arising from chromaffin cells of the adrenal medulla. Closely related tumors of the extra-adrenal paraganglia are termed as paragangliomas (PGLs). Adrenal PCCs are the most common chromaffin tumors, occurring in about 80–85% of cases. The remainder occur as PGLs, which may be classified as branchiomeric, including glomus jugulare and carotid body tumors; intravagal; aorticosympathetic; and visceral-autonomic. These tumors are rare, with an annual reported incidence of two to eight per million population and prevalence in 0.2–0.4% of hypertensive patients. They are characterized by their ability to synthesize, store, and secrete catecholamines although some, especially those of branchiomeric or intravagal origin, are non-functional. Elevated urinary fractionated and plasma free metanephrines have been shown to be sensitive markers of disease and are therefore often used in diagnosis. Although less sensitive for diagnosis, chromogranin A levels are also valuable in following response to therapy and monitoring for recurrence .RET) proto-oncogene; von Hippel–Lindau (VHL) syndrome caused by mutations of the VHL gene; and von Recklinghausen’s disease due to mutations of the neurofibromatosis type 1 (NF1) gene. In addition, mutations in genes encoding succinate dehydrogenase subunits  are associated with familial PGL syndromes , respectively.While most occur as sporadic tumors, about 30–35% is associated with hereditary syndromes. These include multiple endocrine neoplasia type 2 arising from mutations in the rearranged during transfection  remains largely undefined.Currently, the only diagnostic criterion for malignant disease is the presence of metastasis. The proposed incidence of malignant disease ranges from 3 to 36% with generally lower rates of malignancy in PCC compared to PGL. However, rates of malignancy have been reported as high as 50% in patients with Ascribing a role to EBRT in the treatment of malignant PCC/PGL is difficult given limited numbers of patients and reports in the literature. There have been case reports on malignant PCC/PGL in which EBRT was delivered to unknown total doses. In these cases, irradiation was delivered to the primary tumor or metastatic sites with varying symptomatic control and most often with progression of local and distant disease –6. Howev131I-MIBG in non-head-and-neck PCC or PGL , and chromogranin A (CgA) levels within 5–6 months of radiation therapy, total radiation dose and fraction size, radiation technique, symptomatic response, local and distant radiographic response or control, radiation toxicities, additional therapies after treatment, and survival time.A retrospective chart review of endocrinology patients treated at the National Institutes of Health between 1990 and 2012 was performed. Patients were selected if they received EBRT for pathologically confirmed malignant PCC/PGL. Irradiation was performed using either Radiographic response was determined by response evaluation criteria in solid tumors (RECIST) v1.1 based on computed tomography (CT) or magnetic resonance imaging (MRI) reports for a subset of eight patients with 12 lesions with pre and post-treatment imaging available for evaluation ; 1 underwent resection with RFA to liver metastases; 1 underwent resection with systemic yttrium-90 therapy; and 1 underwent resection, systemic chemotherapy , and RFA to iliac crest lesions. Only two patients received biopsy alone prior to EBRT . Eight patients presented with metastases and 16 recurred with metastases an average of 6.2 years (2–264 months) after resection of the primary tumor. One patient had a n = 30), with response of two symptomatic lesions included in analysis lost to follow-up.Patients received EBRT to abdominal, CNS, and bone lesions. Bone lesions were treated with 3D conformal EBRT to a mean dose of 32.6 Gy in 3.9 Gy fractions, CNS tumors to a mean of 30.3 Gy in 2.1 Gy fractions, and abdominal disease to a mean of 54 Gy in 1.8 Gy fractions. The overall mean was 31.8 Gy in 3.3 Gy fractions. Treatments were delivered by SRS to a single bony lesion to 24 Gy; by SRS to a CNS lesion at a dose of 14 Gy; and by FSRT to two abdominal masses to 24.0 and 25.5 Gy in 8.0 and 8.5 Gy fractions, respectively. Of the 47 sites treated, 37 were symptomatic. Of lesions which were symptomatic, improvement in symptoms was reported in 81.1%  and RFA (n = 2). Only three patients died from progressive disease (PD) and mean follow-up time was 52.4 months.Following RT, 13 patients received systemic chemotherapy for an average of 8.7 cycles . Patients were treated with radiopharmaceutical therapy: nine were treated with n = 2) with an average TTP of 22.47 months. For non-target disease, 28.6% of patients (n = 2) achieved complete response (CR), 14.2% had non-PR/non-CR (n = 1), and 71.4% had PD with an average TTP of 13 months. Of the lesions not evaluable by RECIST, local control was achieved in 82.8% of lesions, with 11.4% progressing (n = 4) at an average of 14.8 months. For non-target disease, 16.7% of patients had no distant disease progression while 83.3% progressed at an average of 17.6 months. Overall, local control was observed in 86.7% of patients and progression in 13.3%; distant control observed in 25% of patients and progression in 75%  was achieved in 83.3% and PD in 16.7% (n = 11), 78.5% of symptomatic lesions improved following radiation therapy. Patients with familial syndromes evaluated by RECIST criteria had 84.6% local SD with 15.3% local PD (n = 2) at an average of 13.43 months TTP. They had 92.3% distant PD with 7.6% distant SD and an average TTP of 25.85 months. Of those not evaluated by RECIST criteria, 75% had local control and 25% (n = 1) local progression at 28.63 months TTP. Distant CR was achieved in 33.3% (n = 1) and distant progression occurred in 66.7% with an average TTP of 42.7 months.Of patients with familial syndromes (P = 0.39). Local SD was achieved at an average of 34.75 Gy compared to 31 Gy for those with PD (P = 0.15). Progressive distant disease was observed at an average dose of 33.3 Gy with stable distant disease observed at an average of 36.0 Gy (P = 0.34). In our study, 100% of patients treated with SRS/FSRT achieved local control (n = 4) compared to 85.3% (n = 35) of those treated with standard fractionation.Patients who received 3D conformal or IMRT responded best symptomatically at a higher average dose of 33.7 Gy while average dose for patients without improvement was 31.7 Gy , esophageal (n = 4), and upper gastrointestinal (n = 2) toxicity, as well as grade 2 upper gastrointestinal (n = 4), and grade 3 upper gastrointestinal (n = 1) and esophageal (n = 1) toxicities. There were two reported late toxicities, one esophageal which subsequently resolved and one spinal cord which was permanent in one patient treated by SRS to C3 to 2400 cGy. No patient suffered RTOG acute toxicity grade ≥4 or any RTOG late toxicity grade ≥2.Toxicities from treatment were both acute and late. Patients experienced RTOG grade 1 acute skin (In this retrospective analysis, we evaluated patients with malignant PCC/PGL with primary lesions in the head, neck, and abdomen. Our patients had metastatic lesions to bones, to the abdomen, and to the CNS. This series is the largest on the response of PCC/PGL to EBRT and, unlike many previous case series and case reports, utilizes newer technologies of 3D conformal radiation therapy including IMRT as well as SRS/FSRT. In addition, a subset of our patients was evaluated using standardized radiographic criteria and assessed for biochemical responses to localized radiation therapy. We found an overall symptomatic improvement in 81.1% of patients after radiation therapy regardless of site or radiation technique, with overall local control of 86.7% in patients treated to mean doses of 31.8 Gy in 3.3 Gy fractions by 3D conformal radiation therapy and 21.9 Gy in 10.4 Gy fractions by FSRT.Our study shows a trend of greater symptomatic responses as well as local and distant control in patients who received higher doses of radiation compared to those who received shorter courses of radiation, although statistical significance was not reached possibly limited by the size of our cohort. Given that these treatments were well tolerated, outcomes may have improved with even higher doses of radiation over 40 Gy as suggested in other series and case , 11, 13.In addition, our study shows that most patients will not experience radiographic regression of their disease. This is consistent both with studies of malignant PCC/PGL, but also with extensive literature on EBRT for benign PGL, which has suggested that this is due to the slow growing nature of these tumors . While nIt is also important to consider the site of disease being treated when evaluating dose given by EBRT. While many of our patients with bony metastasis responded well to an average dose of 32.6 Gy, a single patient with a primary malignant glomus jugulare experienced PD by RECIST criteria when treated with 32 Gy. Some studies have indicated that these patients may require doses >60 Gy for durable local control .Fewer of our patients were treated with SRS/FSRT and, of these, only one was symptomatic at presentation. As a newer treatment modality, there is less follow-up time for patients with benign PGL treated with SRS although it has shown promising efficacy in many studies. In our study, durable local control was achieved in a greater proportion of patients treated with SRS/FSRT as compared with standard fractionation, although the number treated using this modality was much smaller. It has been suggested, however, that SRS be restricted in use to smaller tumors <3 cm with a more protracted course of radiation either as 3D conformal EBRT or FSRT for larger lesions .Radiation therapy remains a local therapy, and the majority of patients in this study progressed systemically through their radiation treatments, reinforcing the need for concurrent systemic therapy to control distant metastasis. In spite of this, for patients who were able to be evaluated for biochemical responses after radiation there was a decrease in chromogranin A and catecholamine levels, suggesting a decreased overall disease burden after radiation therapy.As with other reports on the use of EBRT in malignant PCC/PGL, this study is limited by its retrospective nature and the small number of patients treated. However, we have found both 3D conformal EBRT and SRS/FSRT to be effective in controlling symptoms and local progression in patients with both sporadic and familial malignant PCC/PGL. Patients achieved better responses at higher doses in general, although the optimal dose and radiation technique may vary depending on the site treated and size of the lesion. In addition to currently available systemic therapies, EBRT may play a significant role in the control of local disease for these patients.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.


In vitro antivenom efficacy studies were compared to rodent lethality studies to test two Indian snake antivenoms (VINS and BHARAT) against four Sri Lankan snakes. In vitro efficacy was tested at venom concentrations consistent with human envenoming. Efficacy was compared statistically for one batch from each manufacturer where multiple vials were available. In binding studies EC50 for all VINS antivenoms were less than BHARAT for D. russelii . EC50 of both antivenoms was only slighty different for E. carinatus and B. caeruleus. For procoagulant activity neutralisation, the EC50 was lower for VINS compared to BHARAT - 60 μg/mL vs. 176 μg/mL (p < 0.0001) for Russell’s viper and 357 μg/mL vs. 6906μg/mL (p < 0.0001) for Saw-scaled viper. Only VINS antivenom neutralized in vitro neurotoxicity of krait venom. Both antivenoms partially neutralized cobra and didn’t neutralize Russell’s viper neurotoxicity. Lethality studies found no statistically significant difference in ED50 values between VINS and BHARAT antivenoms. VINS antivenoms appeared superior to BHARAT at concentrations equivalent to administering 10 vials antivenom, based on binding and neutralisation studies. Lethality studies were inconsistent suggesting rodent death may not measure relevant efficacy outcomes in humans. Snake envenoming is recognised as a major global health issue with large numbers of deaths and cases of envenoming occurring each year in tropical and sub-tropical regions of the world234650) and median effective dose (ED50) for assessing the efficacy of antivenomsA particular problem with developing antivenoms is testing their efficacy and more importantly the potential efficacy in humans, including appropriate dosing. The World Health Organisation (WHO) recommends the use of median lethal dose  envenoming that the Government requested independent testing of the antivenoms. Treating doctors had observed an increased number of hospital deaths and reactions to the antivenom.Currently all antivenom used in Sri Lanka is supplied from India and there are limited data regarding the efficacy of Indian antivenoms against Sri Lankan snakesin vitro studies to classical lethality studies. In doing so we aimed to test the ability of the antivenoms to neutralise 1) the procoagulant effect of two viper venoms–Russell’s viper (D. russelii) and the saw-scaled viper (Echis carinatus); 2) neurotoxic effect of three snake venoms–common krait (Bungarus caeruleus), common cobra (Naja naja) and Russell’s viper (D. russelii); comparing these to LD50/ED50 studies.The aim of this study was to investigate the efficacy of two different Indian polyvalent antivenoms comparing a range of in vitro experiments were done at venom concentrations measured in cases of human envenoming which was 1.7 μg/ml for D. russelii, 0.5 μg/ml for E. carinatus, 1.4 μg/ml for N. naja and 0.15 μg/ml for B. caeruleus. Multiple batches of antivenoms from both VINS Bioproducts Limited and BHARAT Serum and Vaccines Limited were tested and compared. To statistically compare the efficacy of the two antivenoms one batch from each manufacturer was compared where multiple vials were available–VINS  and BHARAT .All The median dry powder weight (mg) of antivenom, the median percentage of protein per mg of antivenom and the median dry powder weight (mg) of protein per antivenom vial were measured . All bat50) for antivenom binding was the concentration of antivenom that bound 50% of free venom antigens at clinically relevant venom concentrations. The EC50 for both antivenoms are given in 50 values of D. russelii venom for all VINS antivenoms were less than for BHARAT antivenoms, and was 553 μg/mL  for VINS 2010 compared to 1371 μg/mL (95% CI: 956–1965 μg/mL) for BHARAT 2011, which was statistically significantly different (p = 0.016). The EC50 values for N. naja venom of all VINS antivenoms were greater than all BHARAT antivenoms, 336 μg/mL (95% CI: 325–347 μg/mL) for VINS 2010 compared to 70 μg/mL (95% CI: 53–91 μg/mL) for BHARAT 2011 (p < 0.0001). The EC50 values for E. carinatus and B. caeruleus venoms of VINS and BHARAT antivenoms did not appear to differ markedly (B. caeruleus , but not for E. carinatus  .The median effective concentration  compared to 176 μg/mL (95% CI: 149–207 μg/mL) for BHARAT, which was significantly different (p < 0.0001). Much higher concentrations were required by both antivenoms to neutralise the procoagulant effect of E. carinatus despite there being lower concentrations of venom present. (50 against procoagulant activity of E. carinatus for VINS was 357 μg/mL (95% CI: 87–1458 μg/mL) compared to 6906 μg/mL (95% CI: 4859–9817 μg/mL) for BHARAT, which was significantly different (p < 0.0001).VINS antivenoms were able to neutralise the procoagulant activity of both venoms at much lower antivenom concentrations than BHARAT antivenom, based on lower median effective concentrations against procoagulant activity  caused rapid inhibition (t90 = 33.5 min) of nerve-mediated twitches in the chick biventer preparation, while N. naja (N = 4) and D. russelii (N = 3) venoms only caused partial inhibition of nerve-mediated twitches at a concentration of 3 μg/ml (E. carinatus venoms (N = 3) did not cause inhibition of nerve-mediated twitches at any dose. For the increased concentration of 30 μg/ml, N. naja (t90 = 29.5 min) and D. russelii (t90 = 44.0 min) venom did inhibit nerve-mediated twitches  and the median effective dose (ED50) values in mice. LD50 values of D. russelli and B. caeruleus venoms were less than that of E. carinatus and N. naja venom in mouse experiments  and Pakistani krait (B. sindarus) at similar concentrationsAn unusual finding was that BHARAT antivenom had a significantly higher venom antigen binding capacity for A limitation of the study was that only the most recent batches of BHARAT antivenoms were available to be tested and only multiple vials from one batch of each type of antivenom was available. However, the two recent batches of BHARAT were inferior to the recent batches of VINS tested against the important clinical effects of the medically important snakes in Sri Lanka and a direct comparison between batches with multiple vials found VINS to be statistically significantly more efficacious than BHARAT antivenoms. In addition, the variability within these two batches was much greater for BHARAT than for VINS. We have previously shown that expired antivenoms up to 10 years old, formulated as liquids, have lost minimal activity, even after extended periods at room temperaturein vitro studies because the lethality studies are dosed on mouse body weight. This is another reason that lethality studies are problematic because it is difficult to relate μg/g body weight to the amount of venom in human bites. For the binding and in vitro studies we used concentrations measured in human snake envenoming cases. The difference in units did not affect the conclusions of the study because the relative efficacy of the two antivenoms differed in lethality studies compared to the in vitro studies. In addition, the ER50 was also calculated to provide another comparison without units.The units of antivenom differed for the lethality studies compared to the in vivo murine model, evidenced by protein and erythrocytes in urine, but not renal histology or measurements of creatinine. Myotoxicity is only a minor clinical problem in envenoming by all of these snakesD. russelii venom, which is too early to be due to systemic myotoxicity and not consistent with a previous in vivo examination of systemic myotoxicity due to snake venoms in ratsE. carinatus neither of the other haemotoxic effects are important in D. russelii bites.Another limitation was that the study did not test the efficacy of the antivenom against other known venom effects, such as myotoxicity or nephrotoxicityB. caeruleus envenomingD. russelii, and more recent vials had better binding efficacy for B. caeruleus and E. carinatus. The binding efficacy was statistically significantly better for VINS for all snakes except E. carinatus. VINS antivenoms were also more efficacious in neutralising procoagulant effects for D. russelii and E. carinatus, and neurotoxic effects for B. caeruleus. Neither antivenom was able to neutralise the neurotoxic effects of D. russelii venom. Although neurotoxicity occurs in about half of D. russelii bites in Sri Lanka it is rarely life-threateningin vitro efficacy against clinically relevant toxicity.Neurotoxicity is the most important clinical effect in in vitro binding and neutralisation efficacy, efficacy assessed by traditional ED50 testing against lethality in mice, was not statistically significant between the antivenoms for any of the venoms. The relative efficacy of the two antivenoms based on ED50 values was not consistent with any of the testing against important clinical effects in any of the snakes, suggesting that relying on such testing is problematic. The reason for this is that death in animals  could be due to a range of toxicities including some clinically irrelevant toxic effects important in human envenoming. It is entirely possible that post-synaptic neurotoxins or early cardiovascular collapse are major causes of lethality in rodentsin vitro studies of antivenoms against venom effects, and great care should be taken when interpreting in vivo animal models. However, further work is required on other snakes worldwide to confirm our findings for all antivenoms.In contrast to efficacy as assessed by in vitro of D. russelii venom and E. carinatus venom, and the neurotoxic effects of B. caeruleus. In contrast, the lethality studies did not appear to provide as useful an assessment or comparison of the efficacy of the two antivenoms.VINS antivenom appears to be the more efficacious compared to BHARAT antivenom. A dose of 10 vials is sufficient to bind all free venom antigens from these four snakes for venom concentrations found in patients with human envenoming. In addition, this dose was also able to neutralise the procoagulant effects D. russelii), common cobra venom (N. naja), Saw-scaled viper (E. carinatus) and common krait (B. caeruleus) venoms were collected in Sri Lanka. Stock solutions of venom was prepared as 1 mg/mL in 50% Glycerol and stored at −20 °C.Indian polyvalent snake antivenom was obtained from VINS Bioproducts Limited  and from BHARAT Serum and Vaccines Limited . Details of the antivenoms tested are given in D. russelii and E. carinatus venom procoagulant activity studies. Fresh frozen plasma was obtained from the Australian Red Cross and aliquots of 10 mL were thawed at 37 °C. Tetramethylbenzidine (TMB) from Sigma, bovine serum albumin (BSA) from Bovogen, Australia and Streptavidin-conjugated horseradish peroxidase (Streptavidin HRP) from Millipore Chemicon were used for the binding studies. Blocking solution is 0.5% BSA in phosphate buffered saline (PBS). Washing solution is 0.02% TWEEN 20 in PBS. Polyclonal monovalent rabbit IgG to Russell’s viper venom was obtained by injection of rabbit with D. russelii venom followed by purification of the serum on a Protein G-Sepharose column and was carried out at the Western Australian Institute of Medical Research. Polyclonal monovalent rabbit IgG to E. carinatus, N. naja and B. caeruleus venom were obtained by injection of rabbits with the corresponding venoms, followed by purification of the serum on a Protein G-Sepharose column and was carried out at the Faculty of Medicine and Allied sciences, Rajarata University, Sri Lanka. Rabbit IgG antibodies were biotinylated using EZ-Link Sulfo-NHS-LC-Biotin (Pierce # 21335).Bradford reagent  and Bovine Gamma Globulin  were used for protein quantification. Tris-buffered saline (TBS) was used to make up dilutions of antivenom for neutralization of D. russelii venom were undertaken at venom concentrations measured in cases of human envenoming and taken as 1.7 μg/ml, the 97th percentile of venom concentrations in a previous study of 257 patients with pre-antivenom venom concentrations ranging from 0.0033 to 14.8 μg/mLE. carinatus venom the venom concentration used was 0.5 μg/mL based on the maximum concentrations measured in one study of Echis ocellatus envenomingN. naja and B. caeruleus venoms the venom concentrations was again taken as the 97th percentile which was 1.4 μg/ml for N. naja and 0.15 μg/ml for B. caeruleus based on nine envenomed patients from Sri Lanka for each venom. Only one batch of antivenom from each manufacturer were compared statistically -VINS  and BHARAT –because multiple vials were available.Binding and neutralisation studies of Quantification of the protein content in each antivenom was undertaken using the Bradford protein assay methodE. carinatus venom and 0 to 17.1 mg/mL for D. russelii, N. naja and B. caeruleus venom) in blocking solution (0.5% Bovine Serum Albumin in PBS) were incubated with venom  for one hour at room temperature. Unbound venom antigens were detected using a sandwich enzyme immunoassay (EIA) as previously describedD. russelii, 1:25 for E. carinatus, 1:140 for N. naja, 1:15 for B. caeruleus) dilutions in blocking solution. The plates were allowed to stand for 1 h and then washed three times. Next, biotinylated anti-snake venom IgG  was added. After standing for a further hour the plates were washed again. Streptavidin-horseradish peroxidase  was added and left for 1 h. The plate was then washed three times and 100 mL of TMB reagent added and colour allowed to develop for 3.5 min. The reaction was stopped by the addition of 50 mL of 1 M H2SO4. All samples were measured in triplicate, and the averaged absorbance converted to a concentration of the venom of interest by comparison with a standard curve based on serial dilutions of venom.The following antivenom vials were used for antivenom venom binding studies–VINS 1061; VINS 01011/10–11; VINS 01013/10–11; VINS 01023/10–11; VINS 01024/10–11; VINS 01AS11112; VINS 01AS11114; BHARAT A5311006; BHARAT A5311013; BHARAT A5311014. Solutions of increasing concentrations of antivenom  were incubated with 1.7 μg/mL of D. russelii venom or 0.5 μg/mL of E. carinatus venom for 30 min at 37 °C in a 96 well plate. Fresh frozen plasma (100 μl) containing 40 μl of 0.4 M CaCl2/mL and venom-antivenom solution were added simultaneously to each well using a multichannel pipette. After a 5-second shaking step, the optical density at 340 nm was monitored every 30 s for 20 min. The clotting time was defined as the time until the rapid increase in absorbance, as calculated by Gen5 software (supplied with the Biotek ELx808 plate reader).The same antivenom vials used for D. russelii), Common cobra (N. naja) and Indian krait (B. caeruleus) venoms. Chicks  were killed by CO2 inhalation and exsanguination, and the two biventer cervicis muscles were removed from the back of the neck. Each muscle was attached to a wire tissue holder and placed in a 5 mL organ bath filled with physiological salt solution with the following composition (mM): NaCl, 118.4; NaHCO3, 25; glucose, 11.1; KCl, 4.7; MgSO4, 1.2; KH2PO4, 1.2 and CaCl2, 2.5. The organ baths were bubbled with carbogen  and maintained at a temperature of 33–34 °C under a resting tension of 1 g. Motor nerves were indirectly stimulated every 10 s (0.2 ms duration) at supramaximal voltage using a Grass S88 stimulator. The tissues were equilibrated for 10–15 min after which d-tubocurarine (10 μM) was added, and the subsequent abolition of twitches confirmed the selective stimulation of the motor nerves. The tissues were then washed repeatedly until twitch height was restored. Contractile responses to acetylcholine , carbachol , and KCl (40 mM for 30 s) were measured in the absence of stimulationB. caerulus venom had significant neurotoxicity at concentrations seen in human envenoming (3 μg/mL). Neurotoxicity potency was measured as the t90 which is the time required to cause 90% inhibition of the initial twitch height, for a given concentration of venom, expressed as a mean + /− SD. For some venoms, where the twitch height inhibition for a particular venom/treatment did not reach 90%, the time to maximum twitch inhibition observed in all tissues of that treatment/venom group was considered for all the groups for comparison.VINS (01AS11114) and BHARAT (A5311014) antivenom batches were used to investigate the neutralisation of the neurotoxic effects of Russell’s viper (D. russelii (30 μg/mL), N. naja venom (30 μg/mL) or B. caeruleus venom (3 μg/mL) was then added and left in contact with the tissue for 90 min. The antivenom concentration used was based on the manufacturer’s instructions . Experiments were done for both the recommended dose and five times the recommended dose.Antivenom was added to the bath and incubated for 10 min. 50) and the median effective dose (ED50) values in mouse experiments. ICR (Institute of Cancer Research) mice, both sexes, weighing 18–20 g were used for all experiments. The following antivenom vials VINS0101110/11 and Bharat A5311006 were used for lethality studies. For this study, 250 mg/ml solutions of the antivenoms were prepared for venom-neutralization studies which was higher than the manufacturer’s recommended concentration because the recommended concentration was too dilute and therefore too large a volume for administration to miceLethality neutralization studies on antivenoms were tested by calculating the median lethal dose  (2010)50 and ED50 experiments. In all ED50 studies, five times the LD50 of the respective venom was mixed with varying amounts of antivenom and incubated at 37 °C for 30 min before injection. All injections were intravenous to the tail veins and were made to a final constant volume of 250 μl by adding normal saline. In both LD50 and ED50 studies, death/survival rates were recorded for 48 hours. The ED50 values were expressed in μg of antivenom per g body weight of mouse (μg/g) to neutralize the challenge dose of venom. The median effective ratio (ER50) was also calculated using the following formula,Assessment of the LD50 doses given (5 in this study).where n is the number of LDEthical approval for the chick experiments was obtained from the Monash University Animal Ethics Committee 2012/008. All animal experiments were conducted in the Animal House, Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka according to the Council for International Organizations of Medical Sciences (CIOMS) guidelines on animal experimentation50 were fitted by non-linear regression. For the in vitro neurotoxicity data the twitch heights were analysed by one way ANOVA followed by Bonferroni post-hoc tests, and the time to a reduction in twitch heights by Kruskal-Wallis test followed by Dunn’s multiple comparison test. Standard error of the mean (SEM), standard deviations (SD) and 95% confidence intervals (95% CI) were all calculated for parametric and non-parametric data respectively. The difference in protein content between vials from one batch of each of the two antivenoms was compared using an unpaired t-test. Log EC50 values were compared using the extra sum of the squares F test in Prism when comparing one antivenom from each manufacturer. The 95% confidence intervals of the ED50 values were compared to determine if the ED50 values were significantly different. Statistical significance was set at p < 0.05. All analyses and graphics were done in GraphPad Prism version 6.03 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com, except for the calculation of the LD50 and ED50 which was done using the probit methodStandard curves for enzyme immunoassays and calculations of ECHow to cite this article: Maduwage, K. et al. Efficacy of Indian polyvalent snake antivenoms against Sri Lankan snake venoms: lethality studies or clinically focussed in vitro studies. Sci. Rep.6, 26778; doi: 10.1038/srep26778 (2016).


Demyelination is a cardinal feature of multiple sclerosis, but it remains unclear why new lesions form, and whether they can be prevented. Neuropathological evidence suggests that demyelination can occur in the relative absence of lymphocytes, and with distinctive characteristics suggestive of a tissue energy deficit. The objective was to examine an experimental model of the early multiple sclerosis lesion and identify pathogenic mechanisms and opportunities for therapy.Demyelinating lesions were induced in the rat spinal dorsal column by microinjection of lipopolysaccharide, and examined immunohistochemically at different stages of development. The efficacy of treatment with inspired oxygen for 2 days following lesion induction was evaluated.Demyelinating lesions were not centered on the injection site, but rather formed 1 week later at the white–gray matter border, preferentially including the ventral dorsal column watershed. Lesion formation was preceded by a transient early period of hypoxia and increased production of superoxide and nitric oxide. Oligodendrocyte numbers decreased at the site shortly afterward, prior to demyelination. Lesions formed at a site of inherent susceptibility to hypoxia, as revealed by exposure of naive animals to a hypoxic environment. Notably, raising the inspired oxygen  during the hypoxic period significantly reduced or prevented the demyelination.Demyelination characteristic of at least some early multiple sclerosis lesions can arise at a vascular watershed following activation of innate immune mechanisms that provoke hypoxia, and superoxide and nitric oxide formation, all of which can compromise cellular energy sufficiency. Demyelination can be reduced or eliminated by increasing inspired oxygen to alleviate the transient hypoxia. Ann Neurol 2016;79:591–604 The events responsible for the formation of new inflammatory demyelinating lesions in multiple sclerosis (MS) remain unknown.Pathological studies have implicated reactive oxygen and nitrogen speciesLesions have a predilection to form in the periventricular and juxtacortical regions,In addition to a relationship with watershed regions, it has long been apparent that new lesions tend to form around veins,Here, we employ an in vivo model of the early MS lesionSalmonella enterica abortus equi; Sigma‐Aldrich, St Louis, MO) into the right dorsal column at depths of 0.7 and 0.4mm (n = 3 rats per time point); control animals received injections of saline alone (n = 2 per time point). The injection site was marked with charcoal on the dura for subsequent histological localization.In brief, a quarter laminectomy was performed aseptically between the T12 and T13 vertebrae in adult male Sprague Dawley rats , under deep isoflurane anesthesia (2% in oxygen), as described previously.Female Dark Agouti rats (163.7g ± 7.8), were exposed to normobaric hypoxia by substituting oxygen with nitrogen using a ProOx 110 controller  in a purpose‐designed chamber (Biospherix). The hypoxia was gradually introduced by decreasing the oxygen from 21 to 10% over 20 minutes, prior to continuous exposure to 10% oxygen for 6 hours (n = 3) or 24 hours (n = 6). Animals housed in the same chamber, in room air , served as controls.The intravenous probe pimonidazole  was employed to detect tissue hypoxia in both studies, as previously described.To examine the consequence of increasing the concentration of inspired oxygen on LPS‐induced demyelination, animals were randomized into treatment groups exposed to either room air (n = 8) or normobaric hyperoxia  for 2 days following the injection of LPS into the dorsal columns. Animals were housed in a purpose‐built chamber (BioSpherix) for the duration of the treatment, and temperature, oxygen concentration, and carbon dioxide concentration were monitored and controlled throughout. Following treatment, animals were returned to their home cages and maintained at room air until perfusion, 12 days later.All the protocols involving animals were approved by the institutional ethics committee, licensed under the UK Animals (Scientific Procedures) Act of 1986, and conducted according to the ARRIVE guidelines. Animals were provided with food and water ad libitum throughout.All animals were transcardially perfused with rinse solution  followed by paraformaldehyde (4% in 0.15M phosphate buffer) under deep anesthesia (3% isoflurane) after 6 or 24 hours of exposure to 10% oxygen (naive hypoxia study), and at 12 hours or 1, 2, 3, 7, or 14 days after LPS injection (LPS lesion study). To visualize the spinal cord vasculature, animals exposed to 6 hours of 10% oxygen were additionally perfused with the fluorescent carbocyanine lipophilic dye, DiI  after perfusion with rinse solution but prior to perfusion–fixation.Fixed spinal cords were washed in phosphate‐buffered saline (PBS) prior to arrangement in a custom‐built sample holder and immersed in Fomblin perfluoropolyether  to avoid susceptibility artifacts. The holder was positioned in a horizontal bore 9.4T preclinical MRI scanner  equipped with a 33mm‐diameter radiofrequency birdcage volume coil . Images with a spatial resolution of 50 × 50 × 300 µm were acquired using a volumetric gradient echo sequence with the following parameters: echo time = 20 milliseconds, repetition time = 100 milliseconds, flip angle = 35 °, field of view = 28 × 14 × 38.4mm, matrix size = 560 × 280 × 128, number of averages = 12, and a total acquisition time = 12.5 hours.All tissue was postfixed overnight in either 4% paraformaldehyde for cryosections, followed by cryoprotection in 30% sucrose in PBS, or 4% glutaraldehyde in 0.15M phosphate buffer for resin sections, and subsequently processed for examination in frozen or semithin resin sections using standard techniques and the range of antibodies described in the Supplementary Table, as previously described.Tissue labeled using the peroxidase detection system was viewed using an Axiophot light microscope  and photographed with a Nikon D300 camera .Fluorescent images were obtained using a Zeiss LSM5 Pascal confocal microscope, with ×2.5 and ×40 objectives.All analysis and quantification was performed blind using ImageJ . Cells positively labeled with adenomatous polyposis coli (APC), HIF‐1α, and inducible nitric oxide synthase (iNOS) were counted in the spinal dorsal column at the site of injection and expressed as cell density. Pimonidazole labeling was quantified as described previously.t tests between groups at each individual time point. Independent t tests were also used to compare lesion length, maximum cross‐sectional area, and volume between oxygen and room air treatment groups. A Pearson correlation coefficient was used to assess reliability of MRI measures of lesion size in comparison with LFB staining. Probability values of <0.05 were considered statistically significant. All statistical analyses employed SPSS .All data were tested for normality using either the Kolmogorov–Smirnov or the Shapiro–Wilk test, and for homogeneity of variances using the Levene test. Linear regression analysis was used to compare differences between groups for the different immunohistochemical markers, and when significant, further compared using independent To study the relationship between the distribution of pimonidazole labeling and the vasculature, 3‐dimensional (3D) reconstruction was performed on serial sections that were double‐labeled with antipimonidazole and anti–RECA‐1 antibodies using the Reconstruct editor .The unilateral injection of LPS into the dorsal column white matter of the rat spinal cord induced a focal primary demyelinating lesion, as described previously Fig .26, 27 Ap < 0.01; Fig Oligodendrocyte number was assessed using an antibody directed against the marker tumor suppressor APC. The number of oligodendrocytes in the dorsal columns of animals injected with LPS was comparable to saline‐injected controls during the first 2 days, but was significantly decreased at 3 days (p < 0.001) and 2 days (p = 0.014) postinjection, compared with saline‐injected controls. Labeling for pimonidazole in the dorsal column white matter was significantly greater (p < 0.005) than controls at 1 day after LPS injection, and double‐label immunofluorescence revealed colabeling of pimonidazole with carbonic anhydrase 2, a marker of oligodendrocytes, and glial fibrillary acidic protein, a marker of astrocytes. No such labeling was present in saline‐injected controls.Immunoreactivity for pimonidazole was prominent throughout the spinal cord at the level of LPS injection from 12 hours to 2 days after lesion induction, several days prior to the onset of demyelination , 1 day (p = 0.029), and 2 days (p = 0.002) postinjection, compared with saline‐injected controls. HIF‐1α labeling was observed in the cytoplasm and nuclei of cells throughout the dorsal columns and adjacent gray matter 12 hours to 2 days after LPS injection; these were identified as oligodendrocytes based on double label immunofluorescence , 1 day (p < 0.001), and 2 days (p = 0.011) following injection, compared with saline‐injected animals, with the greatest density observed at 1 day , but was prominent in all animals breathing 10% oxygen for 6 hours Fig A or 24 hExposure to 10% oxygen for 6  or 24 hours  reduced, or even absent, in rats treated with 80% oxygen during the first 2 days after LPS injection (n = 11), as determined by the loss of LFB staining , compared with room air controls .Characteristic primary demyelinating lesions were observed at the gray–white matter border in control animals maintained in room air (n = 8) for 14 days following intraspinal LPS injection. In contrast, the area of demyelination was dramatically and significantly  is normally relatively low, and within the white matter it is especially low, such that nearly anoxic values of <1mmHg have been recorded, even in normal tissue  Therapeutically, it is encouraging that oxygen is easily administrated and there is substantial clinical evidence that oxygen administration is generally safe, if delivered at moderate concentration and duration. However, there is a theoretical safety concern that increasing oxygenation could promote oxidative damage, which is already implicated in MS pathology.R.A.D. and A.L.D. designed the study, and were principally responsible for conducting all the experiments, analyzing the data, and writing the manuscript. M.T. conducted the ex vivo MRI experiments and aided with manuscript preparation. X.G. supervised the MRI experiments and aided with manuscript preparation. M.K. and F.L. provided preliminary observations. K.J.S. supervised the project and wrote the manuscript. All authors approved the final manuscript before submission. R.A.D. and A.L.D. are joint first authors.K.J.S. has received research funding for unrelated work from Biogen Idec and Merck Serono.Additional supporting information can be found in the online version of this article.Supporting Information Table 1.Click here for additional data file.


CoA mutase (MUT) is an essential enzyme in propionate catabolism that requires adenosylcobalamin as a cofactor. Almost 250 inherited mutations in the MUT gene are known to cause the devastating disorder methylmalonic aciduria; however, the mechanism of dysfunction of these mutations, more than half of which are missense changes, has not been thoroughly investigated. Here, we examined 23 patient missense mutations covering a spectrum of exonic/structural regions, clinical phenotypes, and ethnic populations in order to determine their influence on protein stability, using two recombinant expression systems and a thermostability assay, and enzymatic function by measuring MUT activity and affinity for its cofactor and substrate. Our data stratify MUT missense mutations into categories of biochemical defects, including (1) reduced protein level due to misfolding, (2) increased thermolability, (3) impaired enzyme activity, and (4) reduced cofactor response in substrate turnover. We further demonstrate the stabilization of wild‐type and thermolabile mutants by chemical chaperones in vitro and in bacterial cells. This in‐depth mutation study illustrates the tools available for MUT enzyme characterization, guides future categorization of further missense mutations, and supports the development of alternative, chaperone‐based therapy for patients not responding to current treatment.Methylmalonyl‐ In humans, this reaction represents an important step in propionate catabolism, funneling metabolites from the breakdown of amino acids , odd‐chain fatty acids, and the side chain of cholesterol into the tricarboxylic acid cycle. The MUT enzyme is highly conserved from bacteria to human and is well studied at the enzymatic propionate into acid precipitable material in intact skin fibroblasts is responsive to supplementation of the culture medium with hydroxocobalamin, are designated mut−; mutants with no residual MUT activity and no response of propionate incorporation to hydroxocobalamin are designated mut0 succinate from [14C]methylmalonyl‐CoA by a modification of earlier described methods methylmalonyl‐CoA , St. Louis, MO, USA; specific activity 7.03 MBq/mmol in assay), and 50–100 μg cell proteins without (holo‐MUT activity) and with  50 μM AdoCbl. To estimate KM for AdoCbl, its concentration was varied between 0.0025 and 50 μM. To estimate KM for methylmalonyl‐CoA, its concentration was varied between 0.01 and 1.0 mM in the presence of 50 μM AdoCbl. Reactions were initiated by the addition of cell lysate, allowed to proceed for 30 min at 37°C, and terminated by addition of 6 μl 5 N KOH . Samples were reincubated for 15 min at 37°C to hydrolyze CoA derivatives, neutralized by adding 5 μl 5 N perchloric acid (Merck), and spiked with 5 μl of 1% solution (w/v) of succinic acid (Merck) in order to visualize succinate during HPLC separation. Samples were centrifuged to remove precipitate, 50 μl of the supernatant was injected into an Amimex HPX‐87H Ion Exclusion column , and [14C]succinate was separated from [14C]methylmalonate by elution with 0.5 mM H2SO4 at 30°C using a flow rate of 0.4 ml/min. Succinate (retention time 17 min) and methylmalonate (11 min) peaks were visualized at 210 nm using a UV detector; 0.2 ml fractions covering the succinate peak were collected and [14C]succinate was quantitated in a Tri‐Carb C1 900TR scintillation spectrometer  with Optiphase HiSafe 2 counting cocktail (PerkinElmer). Protein in cell lysates was determined by the Lowry method. MUT activity was expressed as nanomole succinate formed per minute and milligram of protein. KM values for AdoCbl were determined using Eadie–Hofstee plotting.MUT activity was assayed in crude cell lysates by measuring the production of [Tm), which is the midpoint transition from a folded to an unfolded state, as previously described [Niesen et al., wt protein was assayed in the apo state, or with preincubation of 50 μM AdoCbl (Sigma–Aldrich), or a combination of 50 μM AdoCbl and 500 μM malonyl‐CoA . MCoA was used as a substrate analogue because it has been confirmed to bind to the substrate pocket (PDB: 2XIQ) [Froese et al., Purified homodimeric MUT was assayed for shifts in melting temperature  and milder mut− (n = 16) subtypes, (2) are widely distributed across the gene (exons 2–13), and (3) include representative mutations prevalent in different ethnic populations, for example, African Americans (p.G717V and c.2150G>T) [Worgan et al., SIFT prediction (Table mut− subtype.From the repertoire of known dy Table  with theut− n = 6 subtypeut− n = 6 subtypewww.thesgc.org/MUT). These mutations are located across the entire polypeptide, including the N‐terminal substrate‐binding domain (n = 13), C‐terminal cofactor‐binding domain (n = 9), and interdomain linker (n = 1)  were assessed on their (1) expression level relative to wt protein, as judged by protein amount in total cell lysate, and (2) solubility, as judged by protein amount after affinity purification. We first tested several postinduction growth temperatures (37/26/18/12°C) for wt MUT and observed the highest solubility level with 18°C overnight growth (data not shown). Although all 21 mutants are expressed in E. coli at this temperature  or reduced  protein recovery from the affinity eluant (lanes “E”). This observation, unchanged by a lower postinduction temperature , indicates insoluble mutant protein expression likely due to a global folding defect. In addition to E. coli, we expressed MUT mutants using an immortalized MUT‐deficient patient fibroblast cell line. Western blot , wt MUT exhibited a mean substrate turnover of 20.2 nmol/min/mg protein (Supp. Table S2). All 23 mutants showed decreased enzyme activity, ranging from 0.2% to 85% of wt values , intermediate , and low  levels of residual enzyme activity . All mut0 mutants studied were near the assay detection limit (<2% of wt), consistent with their reported clinical severity, whereas the mut− mutants (2.7%–85% of wt) are distributed into all three levels of activity. The more substantial residual activity of −mut mutations allowed us to further determine their KM values for AdoCbl and methylmalonyl‐CoA. wt MUT had a mean KM for AdoCbl of 4.7 nM (Supp. Table S2), similar to published values [Morrow et al., KM values than wt, to varying degrees  are at least three orders of magnitude higher, indicating a strong KM defect. On the contrary, five proteins had near‐wt KM , suggesting their catalytic impairment is not primarily due to reduced cofactor affinity. No mutants showed large increases in KM for methylmalonyl‐CoA (data not shown), suggesting substrate binding was not defective in these mutants, as has been shown previously [Willard and Rosenberg, MUT catalyzes the isomerization of methylmalonyl‐CoA to succinyl‐CoA via reactive radical intermediates generated from a homolytic bond cleavage of AdoCbl [Banerjee and Ragsdale, E. coli . They had similar retention volumes in size exclusion chromatography as wt MUT [Froese et al., Tm) of each purified protein. As proof of principle, we first interrogated the stability effect of physiological ligands  on the Tm of wt MUT, with the rationale that binding of the cognate ligands should stabilize MUT thereby increasing its Tm. Consistent with this, wt MUT (at 1 μM) exhibits a Tm of 58.2°C in the apo form, which increases in a dose‐dependent manner upon addition of the native‐like ligands , reaching Tm values of 60.8°C (ΔTm = 2.6°C) with AdoCbl (50 μM), and 73.7°C (ΔTm = 15.5°C) with both AdoCbl (50 μM) and MCoA (500 μM) , whereas the larger ΔTm with AdoCbl + MCoA is associated with a substantial conformational change in the N‐terminal domain . Notably, MCoA alone (500 μM) did not increase the Tm of MUT beyond apo levels (data not shown), suggesting an ordered binding mechanism of AdoCbl cofactor first, followed by substrate, in the ternary complex.To test whether the catalytic impairment of certain MUT mutants relates to changes in their structural integrity, we expressed and purified those mutants in large scale that were sufficiently soluble in oli Fig. A, n = 15Tm values of purified mutant proteins in the apo, AdoCbl bound, and AdoCbl + MCoA bound states  responded to AdoCbl + MCoA stabilization with similar Tm shifts as wt, whereas four  did not, suggesting that the latter mutants lost the capacity for native ligand stabilization. In particular, p.T387I showed a concentration‐dependent decrease in Tm when adding AdoCbl alone . The other nine proteins, however, were more thermolabile than wt in their apo forms. Although five  had slightly lower Tm than wt (ΔTm range −2.7 to −1.3) and responded to AdoCbl + MCoA stabilization, four  were substantially more thermolabile than wt  and did not respond to ligand supplementation.We subsequently measured the tes Fig. , to detewt, implicate protein destabilization as a biochemical defect and prompted us to investigate the potential for posttranslational stabilization of mutant proteins by six different osmolytes using DSF and bacterial expression , with the efficacy of stabilization dependent on the osmolyte used . To determine if osmolytes could also stabilize proteins cotranslationally, we expressed several mutants with varying levels of solubility in E. coli and supplemented their growth . Compared with no supplement control, soluble protein yield was improved with glycerol , TMAO (100 mM: p.P615T and p.R694W), and to a lesser extent betaine (100 mM: p.R694W). No improvement for p.L328F was seen, whereas wt protein was very soluble under all conditions, suggesting the rescue could be mutant specific. Together, our data demonstrate the amenability of small exogenous molecules to modulate MUT stability, and support the notion to develop target‐specific “pharmacological chaperones” (PCs).The insolubility and thermolability of a number of MUT mutants, compared with sed Fig. A and B. MUT missense mutations. Earlier small‐scale studies have tested the activity and expression of MUT missense mutants, for example, using the lower eukaryote Saccharomyces cerevisiae (five mutations)  [Janata et al., Our study aimed to establish a genotype–phenotype correlation by characterizing the biochemistry associated with a set of mut0 and mut− subtypes based on the response of a patient cell line to hydroxocobalamin in propionate incorporation [Lempp et al., mut0/mut− subtypes and catalogue the associated biochemical defects, we have further assessed MUT mutations based on stability and catalytic properties as summarized in Table Traditionally, MMA phenotypes are broadly classified into folding and thermolability. Folding defects refer to the aggregation or degradation of translated polypeptides, thereby reducing the steady‐state protein. These mutants displayed drastically reduced soluble protein yield, as exemplified by p.P615T/L. Four other mutants, two from each mut subtype , also fit this category having reduced detectable protein in one of the two expression systems. A few mutants displayed only slightly decreased soluble E. coli expression, such as p.G717V and p.R694W, consistent with a previous report [Crane and Ledley, folding category. Thermolabile mutants had near‐wt levels of recombinant expression and solubility, reflecting near‐native folding, but increased temperature sensitivity. Four mutations in this study fell into the thermolabile category .We evaluated mutants on two stability criteria, folding and thermolabile mutants involve amino acid changes that (1) remove a conformationally restricted proline residue , (2) cause steric clashes with a bulky side chain , or (3) disrupt the natively charged/polar  or nonpolar  environment. Furthermore, stability mutations are distributed across the entire polypeptide, indicating that all domains of the protein contribute to its intrinsic stability.Inspection of the MUT crystal structure shows that both catalytic and KM mutants. Catalytic mutants had low residual activity (<5% of wt) despite detectable eukaryotic expression and constituted the largest category of mutants , one was thermolabile with a catalytic dysfunction (p.R694L), and three met the criteria of folding and catalytic defects . In addition, three mutations were termed unclear  because in our assays they lack definitive aberration in stability and enzyme kinetics, showed detectable protein by Western blot, intermediate enzyme activity, and only moderate KM change.It is of note that our classification scheme is not aimed at Tm shift from MCoA + AdoCbl but not MCoA alone; (2) potential formation of a supramolecular complex with the GTPase MMAA and adenosyltransferase MMAB [Froese and Gravel, Future studies should aim to address the lesser characterized biochemical aspects of MUT and possible mutational effects, which are beyond the scope of this study. These include (1) possible allostery between substrate and cofactor binding, which could potentially explain the MUT genotypes is to help prognosticate the course of the disease. As expected, the characterized mut0 mutants harbor folding or catalytic defects, resulting in very low or undetectable residual enzyme activities consistent with their predominance in neonatal onset patients with severe long‐term complications. On the contrary, the characterized mut− mutants can present in any one, or combinations, of the four biochemical defects defined here. We postulate that mutations that do not interfere with protein stability and have less of a catalytic penalty would result in milder disease . Similar to other metabolic diseases where an activity threshold of ∼5%–10% of wt is sufficient to ameliorate disease [Leinekugel et al., mut− patients often show a later onset and less long‐term complications, presumably due to the intermediate level of residual MUT activity.A clinical application for our biochemical categorization of −mut subtype, benefit from treatment with high doses of hydroxocobalamin is controversial. Early publications documented no response [Wilcken et al., 4) of phenylalanine hydroxylase (PAH) [Zurfluh et al., mut‐type MMA, a clinical response to treatment is usually defined as reduced plasma or urinary concentration of methylmalonic acid. Although difficult to measure because of its broad variation in body fluids [Thompson and Chalmers, 4 on mutant PAH. Our results also predict that patients harboring a KM defect  are more likely to benefit from cobalamin treatment based on the assumption that increased hydroxocobalamin plasma levels result in increased cellular AdoCbl production because, in our enzyme assay, these mutants exhibited high activity levels in the presence of high AdoCbl concentration. Nevertheless, hydroxocobalamin treatment may only benefit a small subset of patients with intermediate to high residual activity, if any, outlining an imperative to search for alternative therapies.Whether any MUT‐deficient patient, including those of the Our stability data add MUT to the growing list of metabolic enzymes in which mutation‐induced protein destabilization is widely believed to play a causative role in disease pathogenesis [Gregersen et al., catalytic, KM mutations, without stability defects, may also benefit from PCs that potentially increase residual activity, acting as “enzymatic activators.” Altogether, the stage is set for a systematic, nonbiased screening regime in the search for PC molecules targeting MUT, and our established DSF assay may provide a good starting point for this screening.Alternatively, target‐specific PCs could be developed for MUT that, unlike chemical chaperones, exert a direct stabilization effect by binding to the affected protein, potentially allowing lower, clinically tolerable dosage concentrations. PCs have shown promise in several lysosomal storage diseases, entering early‐stage clinical trials for Gaucher, Tay‐Sachs, and Fabry diseases [Boyd et al., Disclaimer: Supplementary materials have been peer‐reviewed but not copyedited.Figure S1. Structural view of the amino acid environment for each mutation in this study. For each panel, the amino acid of interest is coloured cyan. Secondary structure elements are coloured cyan for the N‐terminal substrate binding domain, yellow for inter‐domain linker, and magenta for C‐terminal cobalamin binding domain. For panels a, b, d, e, i and l, malonyl‐CoA is shown as sticks (yellow carbon atoms). For panels d, k, p, q, s and t, adenosylcobalamin is shown as sticks (black carbon atoms). For panels a, g, k and m, the neighboring subunit in the MUT dimer is shown as black cartoon. For panels c, h, m, o and q, amino acids surrounding the site of interest are also shown in spheres, to highlight the tight steric packing. Where applicable, hydrogen bonds are shown as dashed lines (distance in angstrom). An interactive ver‐sion of this structural representation is available at www.thesgc.org/MUT.Figure S2. Enzyme activities of MUT wt and mutants in decreasing order. Each bar represents the mean of at least two replicate experiments (error bars depict SEM). Black bars indicate high , dotted bars intermediate  and white bars low  levels of ac‐tivityFigure S3. Ligand‐dependent thermal denaturation of MUT. wt MUT is stabilized with increasing concentrations of AdoCbl (A) and malonyl‐CoA (B). Mutant p.T387I is destabilized with increasing AdoCbl concentrations (C).Figure S4. Substrate/cofactor‐induced conformational changes in MUT. A. Superposition of MUT structures in the apo and AdoCbl‐bound (holo) states shows modest rearrangement in the C‐terminal domain (boxed) by the binding of AdoCbl alone. B. Superposition of MUT structures in the holo and ternary (AdoCbl and MCoA bound) states reveals substantial conformational changes in the N‐terminal domain (boxed) by the additional binding of MCoA. Ligands are shown in sticks .Figure S5. Concentrations‐dependent thermal stabilization in wild‐type and mutant MUT upon addition of chemical chaperones: A. proline, B. glycerol, C. betaine, D. sorbitol, E. TMAO, F. sucrose.Figure S6. SDS‐PAGE of test purification for wild‐type, p.A191E, p.L328F, p.G426R, p.P615T and p.R694W after co‐translational expression in the presence of chemical osmolytes. Glycerol (at 1% and 5%), TMAO and betaine at 100 mM were tested. For clarity, only the relevant gel section is shown. CC: chemical chaperone; TLC: total cell lysate; E: eluant after affinity purification; marker on left: sizes in kDa.Table S1. Primers used for site‐directed mutagenesis of pTracer‐MUT wt and pNIC‐MUT wt constructsTable S2. Methylmalonyl‐CoA mutase (MUT) activities and KM values for the cofactor, ade‐nosylcobalamin (AdoCbl)Click here for additional data file.


BMI) and gestational weight gain (GWG) on offspring body composition. In this prospective cohort study, offspring body composition at 6 years of age was obtained through air displacement plethysmography. Linear regression was used to obtain crude and adjusted coefficients. Information regarding offspring body composition and maternal pre‐pregnancy BMI was available for 3156 children and on offspring body composition and GWG for 3129 children. There was a direct association of maternal pre‐pregnancy BMI and GWG with offspring's fat mass (FM), fat‐free mass (FFM), fat mass index (FMI), fat‐free mass index (FFMI) and body fat percent (BF%) in crude and adjusted analyses. After adjustment for co‐variables, for each kg m−2 of maternal pre‐pregnancy BMI increase, there was a mean increment of 0.13 kg in the offspring FFM, 0.06 kg m−2 in FFMI, 0.11 kg in FM, 0.07 kg m−2 in FMI and 0.18% in BF%. For each kilogram of maternal GWG increase, there was a mean increment of 0.08 kg in offspring's FM, 0.05 kg m−2 in FMI, 0.04 kg in FFM, 0.01 kg m−2 in FFMI and 0.18 % in BF%. Mothers with a higher pre‐pregnancy BMI or GWG tend to have children with greater adiposity at age 6 years. Fetal overnutrition is more likely among mothers with greater BMI during pregnancy; as a consequence, it can accelerate the childhood obesity epidemic.This study aims to investigate the effect of maternal pre‐pregnancy body mass index ( The increase in overweight and obesity prevalence observed in low‐ and middle ‐income countries has been recognised as an important public health problem  during childhood growth , a third population‐based birth cohort study was initiated in 2004. All mothers who resided in the urban area of Pelotas or in the adjacent neighbourhood of Jardim América  were approached for recruitment and interview within 24 h of delivery. All five maternity hospitals were visited daily and the mothers were interviewed via a standardised questionnaire by trained fieldworkers . Demographic, socio‐economic, reproductive, behavioural, prenatal care, gestational and pre‐gestational morbidities, and newborn characteristics were investigated (Santos 2). Maternal weight at the beginning of pregnancy was extracted from the prenatal record or, when absent, by self‐report. Maternal height was measured in the household, at the first follow‐up visit, 3 months after delivery. BMI was classified into four categories: underweight (<18.5 kg m−2), normal weight (18.5–24.9 kg m−2), overweight (25.0–29.9 kg m−2) and obese (>30.0 kg m−2)  at the beginning of pregnancy divided by height in square meters  guidelines as inadequate, adequate or excessive. Women in the underweight category should have a goal of 12.5–18.0 kg weight gain during pregnancy; those with normal BMI, 11.5–16.0 kg; overweight, 7.0–11.5 kg; and obese, 5.0–9.0 kg. Adequate weight gain for preterm and post‐term pregnancies was calculated using the expected weekly weight gain , schooling at birth, skin colour , smoking and alcohol consumption during the pregnancy, parity, age and history of arterial hypertension and diabetes mellitus.−2) were calculated based on FM in kilograms or FFM in kilograms and height in square meters (m2). The indexed parameters have the advantage of taking into account the child height, which improves the sensitivity to detect changes in body composition measurements. Same‐height children can have different percentages of FM due to different absolute amounts of FM but equivalent amounts of FFM or different absolute amounts of FFM and same amounts of FM . Descriptive statistics were calculated for all basic variables that compared children included in the analyses with those lost to follow‐up or with missing information on body composition and/or maternal pre‐pregnancy BMI and GWG. The chi‐square test and analysis of variance (ANOVA) assessed differences in characteristics between these groups. For comparison of offspring mean FM, FFM and BF%, according to maternal pre‐pregnancy BMI and GWG, the one‐way ANOVA test for homogeneous variances and the ANOVA test for linear trend were used. Linear regression was used to obtain crude and adjusted coefficients on offspring FM, FMI, FFM, FFMI and BF%. Adjusted analyses were conducted according to a hierarchical analytical model constructed in three levels. Family income, maternal schooling, maternal skin colour, maternal age, parity, pre‐gestational arterial hypertension, pre‐gestational diabetes and pre‐pregnancy BMI composed the first level. The second level comprised maternal characteristics  as well as child characteristics at birth . In the third level, there were the outcomes of interest: offspring FM, FMI, FFM, FFMI or BF%, at 6 years of age. The effect of the maternal pre‐pregnancy BMI was adjusted for variables of the first level. The effect of GWG was controlled for variables of the first and second levels. Interaction between pre‐pregnancy BMI and maternal schooling over offspring FM, FMI and BF% was identified. The adjustment to the interaction term was included in the final multivariable model by using the MFPIgen command , to select the confounder model at the 5% significance level and to identify interaction between two variables  greater than the FMI of children from mothers who gained sufficient weight  was similar to that reported by Berkowitz et al. (β = 0.27 kg) at 6 years of age and by Lawlor et al. (β = 0.25 kg) among children aged 9–11 years. Similarly, with regard to BF%, the present results are in agreement with the reports of three studies, two of infants  and adjusted (β = 0.10 kg) analyses were identical to the results (β = 0.10 kg) of the present study. The correlation between maternal pre‐pregnancy BMI and offspring FFMI is different from the results reported by Gale et al.  of the cohort participants at 6 years of age, thus allowing the extrapolation of the present results to the population of Pelotas as well as to other populations with similar economic and socio‐cultural contexts. Anthropometric measurements were carried out with standard methodology and trained fieldworkers performed the equipment management. Also, one of the major strengths of the study was that children's body composition was measured using a reference method. A limitation of the study was the use of maternal self‐reported weight before pregnancy or the record of weight at the first prenatal visit, which can lead to some degree of classification error in the exposures. Although self‐reported maternal weight has a high correlation with weight measurement .Maternal gestational as well as pre‐gestational diabetes mellitus is one of the known risk factors for offspring adiposity and is also associated with maternal obesity. According to the intrauterine overnutrition hypothesis, high maternal plasma concentrations of glucose, free fatty acids and amino acids are more likely among mothers with greater BMI during pregnancy, and consequently, fetal overnutrition . From 2009 to 2013, the Wellcome Trust supported the 2004 birth cohort study. The World Health Organization, National Support Program for Centers of Excellence (PRONEX), Brazilian National Research Council (CNPq), Brazilian Ministry of Health, and Children's Pastorate supported previous phases of the study. I. S. Santos and A. Matijasevich receive research support from the National Council for Scientific and Technological Development (CNPq), Brazil.The authors declare that they have no conflicts of interest.HC and ISS conceived the paper, conducted the analysis and wrote the manuscript. AM contributed to the design of the study. All authors revised and approved the final version of the paper for publication.


Furthermore, an apparent similarity has                    been pointed out between the fat face illusion and the Jastrow illusion. Recent                    studies have suggested the importance of facial contours and the role of                    basic-level processing of faces. In the present study, we directly compared the                    typical Jastrow illusion and fat face illusion in humans and chimpanzees using                    the same task. Both humans and chimpanzees clearly showed the Jastrow illusion,                    but only humans perceived the face at the bottom as fatter than the top.                    Although further examination is necessary, these results might reflect different                    processing levels of faces between the two species.When two identical faces are aligned vertically, humans readily perceive the face                    at the bottom to be fatter than the top one. This phenomenon is called the                                                     The second was reported by fat face            illusion: When two identical faces are aligned vertically, the face at the bottom is            perceived to be fatter than the one on top . Recently, several types of illusions concerning face width or size have been reported.            The first was demonstrated by e on top . Kitaokae on top  and name        As         narrower or thinner stimulus of the two. The            narrower or thinner stimulus appeared randomly at the top or bottom position from trial            to trial. Chimpanzees received a food reward  for every correct            choice, while humans were not given any feedback. For chimpanzees, when the width of the            stimuli was the same, the response was randomly reinforced with the probability of 50%.            Initially each chimpanzee was given baseline training for each condition and then 16            test sessions , while each human participant received 2 sessions  for each condition.To directly compare the Jastrow and fat face illusions, we prepared a simple            discrimination task as presented in         t(15) = 9.44,            p < .001,            r = .93; humans,            t(7) = 12.16,            p < .001,            r = .98), while they showed neither underestimation nor            overestimation with the length of the rectangles  = .14, ns,            p = .888, r = .04;            humans, t(7) = 1.99, ns,            p = .086, r = .60).            More interestingly, chimpanzees and humans showed clear differences when they judged the            width of the faces. Humans showed a very strong fat face illusion for            both human and chimpanzee faces  = 5.39, p = .001,                r = .90; chimpanzee face,            t(7) = 6.06,            p < .001,            r = .92). The estimated size of the illusion was 3.2%,            comparable to a previous study  = .40, ns,            p = .697, r = .09;            chimpanzee face, t(19) = 1.51, ns,            p = .148, r = .33).                The results are shown in tudy 4%; . However        This is the first reported study to demonstrate the Jastrow illusion in chimpanzees.            However, they showed no evidence for the fat face illusion. One possibility is the            difference in task procedure between species; chimpanzees were differentially reinforced            but not humans. This procedural difference might have affected the present results.            However, although this issue should be examined in the future, chimpanzees did show the            Jastrow illusion under the identical task for the fat face illusion, suggesting that the            current task is sufficient for studying the visual illusion in nonhuman primates.            Furthermore, differential-reinforcement tasks have been frequently and successfully used            for nonhuman animals e.g., . At best


Although recent studies have suggested that adherence to antihypertensive treatment reduced stroke incidence, the relationship of adherence to antihypertensives with stroke severity has not been studied. This study attempted to know whether nonadherence before stroke is associated with initial severity of acute ischemic stroke.Consecutive patients with acute ischemic stroke were identified in Soonchunhyang University Hospital from Mar 2005 to Aug 2014, excluding the cases without hypertension or information of antihypertensive adherence. We compared the mean of National Institute of Health Stroke Scale (NIHSS) score between adherence groups and insufficient medication group, and additionally in each stroke subtype. Multiple linear regression model was established for initial NIHSS score adjusting alleged factors linked to stroke severity.VS 5.4 ± 5.7, P = .11). In large artery atherosclerosis (LAA) and small vessel occlusion (SVO), initial NIHSS score were significantly higher in insufficient medication group . In multiple linear regression model, insufficient medication to antihypertensives had a significant effect on NIHSS score  after adjusting covariates.Initial NIHSS score were higher in insufficient medication group than adherence group (6.5 ± 7.2 Insufficient medication of antihypertensives before stroke was independently associated with the severity of acute ischemic stroke. Further studies with prospective designs are warranted to evaluate clinical implication of adherence to antihypertensives for ischemic stroke. Benefits of blood pressure lowering for primary stroke risk reduction are broadly consistent across a range of different population –4. RecenAdherence to antihypertensive is closely related to the time within therapeutic range, and may influence on the cardiovascular prognosis . The assThe initial stroke severity has been reported as the most important predictor for poststroke outcome –16, and This study aimed to investigate whether nonadherence to antihypertensives prior to acute ischemic stroke is associated with initial stroke severity in patients with hypertension.A total 2335 consecutive patients with first acute (<7 days) ischemic stroke or imaging-positive transient ischemic attack were registered in Soonchunhyang University Hospital from Mar 2005 through Aug 2014. This web-based registry contains clinical characteristics, laboratory/imaging findings, and cardiovascular outcomes after discharge . The stun = 93, 5.6 %).Data were prospectively collected including history of hypertension, medication history, vascular risk factors, laboratory findings, stroke subtypes, initial National Institute of Health Stroke Scale (NIHSS) Score. Adherence to antihypertensives during the last one month were captured by patient’s or caregiver’s information, and were categorized into two groups: adherence (regular medication) and insufficient medication . The patients who took medications more than 80 % of the last one month were categorized as regular medication. The cases with missing data for adherence to antihypertensives prior to acute ischemic stroke were excluded (n = 29). Finally, 1538 cases were analyzed.Stroke subtypes were determined by extensive workups including magnetic resonance imaging/angiography, echocardiography, and Holter monitoring in all patients: large artery atherosclerosis (LAA), small vessel occlusion (SVO), cardioembolism (CE), and undetermined etiology (SUE). Stroke with other determined etiology were excluded .Categorical variables were reported as frequency and continuous variables as mean ± standard deviation. Chi-square test was used to determine the relationship with categorical variables, and student T-test to compare the means of continuous variables between adherence and insufficient medication group. The difference of NIHSS score between adherence group and insufficient medication group were determined by T-test in each stroke subtype. Multiple linear regression model was established for initial NIHSS score adjusting covariates which have an influence on initial stroke severity in univariate analysis and previous reports. Statistical significance was defined as n = 54), irregular medication before acute stroke (n = 164), and diagnosed at admission (n = 88). The average age were 69.6 ± 11.5 in adherence group while those in insufficient medication group were 66.1 ± 12.2 (P < .001). The fraction of insufficient medication group were larger in female than male . Insufficient medication group had higher initial NIHSS score than adherence group . The baseline characteristics between adherence and insufficient medication group were depicted in Table The patients who had taken antihypertensives regularly before stroke (adherence group) were 1232 (80.1 %). Insufficient medication group had 306 (19.9 %) patients: diagnosed but no medication (n = 17). NIHSS score were significantly higher in insufficient medication group in LAA and SVO: 6.1 ± 6.5 VS 4.4 ± 4.4, P = .004 for LAA; 3.8 ± 3.5 VS 2.7 ± 1.8, P = .014 for SVO. There was a tendency of higher NIHSS score of insufficient medication group in CE  and SUE .Mean NIHSS scores among stroke subtypes were compared in Table F = 18.716, P < .001). The strongest factor which influence on NIHSS score was atrial fibrillation . Age , initial systolic blood pressure  and previous stroke history  were also significant. Insufficient medication to antihypertensives had an independent effect on NIHSS score  . The fitness of regression model was good , and may be induced by increased sympathoadrenal tone, direct brain injury, systemic condition or stress response . The difVS 5.6 %, P = .121). Moreover, individual adherences to medications were not different across vascular risk factors [F = 18.716, P < .001), and there was no multicollinearity between variables in our regression model (VIF <2). Finally, in our study, insufficient medication patients had more smokers and higher serum cholesterol level. Although the association between insufficient medication and stroke severity was significant after adjusting these variables, the social habit and healthy conditions should be considered as influencing factors on nonadherence in Korean.Our study has several limitations. First, the data of insufficient medication were captured only for antihypertensives, but not for antiplatlets, antidiabetics or lipid lowering agents. Prestroke antiplatelets may be associated with stroke severity , 40. How factors . Second,In conclusion, our study in the patients with acute ischemic stroke, insufficient medication to antihypertensives before stroke was independently associated with stroke severity. Further prospective cohort are warranted to evaluate clinical implication of adherence to medications for prevention of cardiovascular disease.


Postoperative nausea and vomiting (PONV) is a displeasing experience that distresses surgical patients during the first 24 h after a surgical procedure. The incidence of postoperative nausea occurs in about 50%, the incidence of postoperative vomiting is about 30%, and in high-risk patients, the PONV rate could be as high as 80%. Therefore, the study design of this single arm, non-randomized, pilot study assessed the efficacy and safety profile of a triple therapy combination with palonosetron, dexamethasone, and promethazine to prevent PONV in patients undergoing craniotomies under general anesthesia.The research protocol was approved by the institutional review board and 40 subjects were provided written informed consent. At induction of anesthesia, a triple therapy of palonosetron 0.075 mg IV, dexamethasone 10 mg IV, and promethazine 25 mg IV was given as PONV prophylaxis. After surgery, subjects were transferred to the surgical intensive care unit or post anesthesia care unit as clinically indicated. Ondansetron 4 mg IV was administered as primary rescue medication to subjects with PONV symptoms. PONV was assessed and collected every 24 h for 5 days via direct interview and/or medical charts review.n = 12). The incidence of nausea and emesis 24 h after surgery was 30% (n = 12) and 7.5% (n = 3), respectively. The mean time to first emetic episode, first rescue, and first significant nausea was 31.3 (±33.6), 15.1 (±25.8), and 21.1 (±25.4) hours, respectively. The overall incidence of nausea and vomiting after 24–120 h period after surgery was 30% (n = 12). The percentage of subjects without emesis episodes over 24–120 h postoperatively was 70% (n = 28). No subjects presented a prolonged QTc interval ≥500 ms before and/or after surgery.The overall incidence of PONV during the first 24 h after surgery was 30% .NCT02635828 ( Postoperative nausea and vomiting (PONV) is a displeasing experience that distresses surgical patients during the first 24 h after a surgical procedure –8. The iAccording to the Consensus Guidelines for the Management of Postoperative Nausea and Vomiting, a combination of antiemetic medications is recommended as the most appropriate regimen for patients with moderate and high-risk factors for PONV . Early PThe Apfel Simplified Risk Score is one of the two most commonly used and effective risk scores to assess inpatient risk of developing PONV when undergoing inhaled anesthesia. The Apfel Simplified Risk Score is based on four predictors: female gender, non-smoking status, history of PONV and/or motion sickness, and use of postoperative opioids. The incidence of PONV with the presence of 0, 1, 2, 3, and 4 risk factors increases drastically, by 10, 20, 40, 60, and 80%, respectively. Patients with 0–1 are considered low risk, those patients with 2 risk factors fall into “medium risk”; and those patients with 3 or more risk factors are considered to be “high risk” ] receptors and exhibits antiemetic and sedative properties. IV infusion of promethazine typically relieves PONV after 5 min and lasts for 2–6 h , 15. HowPalonosetron is the latest serotonin receptor antagonist , 16, 17.QTc prolongation may suggest an inherited predisposition to sudden cardiac death. It may also uncover an increased vulnerability to the development of life threatening ventricular tachycardia in patients being considered for treatment with a known QT-prolonging drug –24. The Therefore, the study design of this single arm, non-randomized, pilot study assessed the efficacy and safety profile of a triple therapy combination with palonosetron, dexamethasone, and promethazine to prevent PONV in patients undergoing craniotomies under general anesthesia.clinicaltrial.gov during enrollment period because it was not part of our institutional standard operation procedures; thus, it was registered after enrollment completion . The study’s inclusion criteria consisted of adult patients, 18–85 years, undergoing neurological surgery (opening of the cranium and dura mater) requiring at least 1 h of general anesthesia and at least 72 h of postoperative hospitalization. These subjects were assessed preoperatively using the Apfel Simplified Risk Score. Exclusion criteria consisted of subject with prisoner or mentally ill status, history of alcohol or drug abuse, hypersensitivity to any of the medications administered in the protocol, female subjects who are pregnant or breastfeeding, history of vomiting or severe nausea 24 h prior to a procedure, history of treatment with any drug or treatment within last 24 h prior to start of the treatment, and/or subjects who have participated in any other clinical study.The research protocol was approved by the institutional review board and 40 subjects provided written informed consent prior to any research procedures at The Ohio State University Wexner Medical Center and completed the study. The protocol was not registered in An electrocardiogram (ECG) was obtained at baseline, 24 and 120 h after surgery, if the subject remained hospitalized. At induction of anesthesia, a triple therapy of palonosetron 0.075 mg IV, dexamethasone 10 mg IV, and promethazine 25 mg IV was given as PONV prophylaxis.−1 was administered before tracheal intubation. General anesthesia was preserved with volatile anesthetics  and clinical judgment regulated titration concentrations. The analgesia during anesthesia maintenance was done with Fentanyl boluses of 0.5–2.0 μg/kg−1 IV and/or hydromorphone boluses 0.5 mg IV. In order to reverse the residual neuromuscular block at the end of the procedure, neostigmine and glycopyrrolate were used.The general anesthesia regimen recommended began with premedication of midazolam 1–2 mg IV promptly before the subject was transferred to the operating room. Propofol 1–2 mg/kg IV and fentanyl 0.75–1.5 μg/kg IV were used to induce anesthesia. Rocuronium 0.6–1.2 mg/kgAfter surgery, subjects were transferred to the surgical intensive care unit (SICU) or post anesthesia care unit (PACU) as clinically indicated. Ondansetron 4 mg IV was administered as primary rescue medication to subjects with PONV symptoms. Subjects who did not respond to this initial treatment were given a second line of therapy. PONV was assessed and collected every 24 h for 5 days via direct interview and/or medical charts review. Episodes of nausea, vomiting, and administration of rescue therapy for either nausea or vomiting were recorded, in addition to the severity of each nauseous or emetic episode. Nausea was rated by the patients utilizing a verbal response scale (0–10), with 0 being no nausea at all and 10 being severe nausea. Vomiting was evaluated by the investigator or nursing staff numerically as either 0 (no vomiting), 1 (mild vomiting), 2 (moderate vomiting), or 3 (severe vomiting). Following the first 24 h after administration of the prophylactic triple therapy, an ECG was conducted and standard of care blood was drawn for safety analysis. If the patient was discharged before the 5-day time period, the patient was then contacted via phone call by research personnel to complete PONV and safety assessments.p ≤ 0.05.Descriptive statistics are reported as mean ± SE and median (range) or total number and percentage. Binomial test were performed to compare the Apfel risk factors between our study and literature. These data were analyzed using Statistical Analysis Software, version 9.3 . Significance was accepted if n = 5), consent withdrawal (n = 0), and others (n = 9) . The incidence of nausea and emesis 24 h after surgery was 30% (n = 12) and 7.5% (n = 3), respectively. Of those patients who experienced nausea during the first 24 h after surgery, the median worst nausea score was 6 . The median severity of vomiting was 2 , corresponding to moderate severity. Rescue medication during the first 24 h was used on 12 patients (30%) , 15.1 (±25.8), and 21.1 (±25.4) hours, respectively (Table n = 4), nausea and vomiting occurred in 10% (n = 4) and 0% (n = 0), respectively. The worst nausea and vomiting score was 9  and 3 , respectively (Table In the course of the early PONV (0–2 h), the incidence of PONV was 10% n = , nausea n = 12). The percentage of subjects without emesis episodes over 24–120 h postoperatively was 70% (n = 28). Of those subjects who experienced nausea during 24–120 h after surgery, the median severity was 6 . The median severity of vomiting was 2.5 , corresponding to moderate severity  and 13.9% (n = 5), respectively. The incidence of a change in QTc interval larger than 60 ms 24 h after surgery was 2.8% (n = 1). The incidence of prolonged QTc interval ≥450 ms 120 h post-surgery was 19.2% (n = 5). The percentage of subjects who presented a change in QTc interval larger than 30 and 60 ms were 11.5% (n = 3) and 0%, respectively , bradycardia (4%), headache (3%), and constipation (2%) . The resIn our study, only 13.9% of patients presented a prolonged QTc interval of ≥450 ms and 0% of patients presented a prolonged QTc interval ≥500 ms. Since prolongation of QTc interval was not our primary objective, more studies with a proper power analysis should be performed to conclude that palonosetron is a low risk for QTc prolongation.Latz et al. presented a prospective study that evaluated the incidence and risk factors of PONV in 229 patients undergoing craniotomy . It founChun et al. published a randomized, double-blind, placebo-controlled trial that proved the efficacy of palonosetron as PONV prophylaxis in 204 patients . In thisMansour designed a double-blind, active controlled study with 150 patients comparing the use of dexamethasone alone, dexamethasone–metoclopramide therapy, or dexamethasone–palonosetron combination therapy . The dexMin et al. presented a retrospective study that analyzed a database of 81 patients who underwent general anesthesia. The main focus of this study was to evaluate changes in QTc interval throughout different time points in surgery . The results demonstrated that the incidence of QTc prolongation ≥500 ms was higher in patients who received 0.075 mg of palonosetron intravenously than those who did not during general anesthesia . NonetheA study by Morganroth et al. included 221 healthy subjects who were assigned to receive one of the following five treatments: placebo, palonosetron , or moxifloxacin (400 mg) in order to study the effects of palonosetron on QTc interval . All thrOur study also presented certain limitations that should be mentioned. To begin with, this study enrolled 40 subjects who received study medication and completed all the assessments, but the study was designed without a control group; therefore, we were not able to compare the results from this regimen. In addition, headache, constipation, fatigue, and dizziness are the most common side effects of palonosetron; however, both side effects are also expected postoperative symptoms after craniotomies .n = 1). In conclusion, this study had demonstrated that the prophylactic regimen used could be an adequate regimen to prevent PONV in subjects undergoing craniotomies under general anesthesia. However, since prolongation of QTc interval was not our primary objective, and we did not have a control group; more studies with a proper power analysis and combined therapy should be performed to demonstrate the efficacy of this regimen and that palonosetron is a low risk for QTc prolongation.The primary objective of this study was to evaluate the efficacy of triple therapy with palonosetron, dexamethasone, and promethazine for the prevention of PONV in subjects undergoing craniotomies. Our results showed a PONV incidence of 30% and an accumulative incidence of nausea and vomiting of 3 (7.5%). In addition, our data had shown the incidence of a change in QTc interval larger than 60 ms 24 h after surgery was 2.8% (SB and EP designed the study, developed the methodology, and wrote the manuscript. AU and MA developed the methodology, collected the data, performed the analysis, and wrote the manuscript. GC and VY performed the analysis and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.