Renal angiomyolipoma (AML) is a common benign tumor of the kidney. The main complication of AML is retroperitoneal hemorrhage caused by AML rupture, which can be severe and life threatening. The risk of AML rupture used to be determined by tumor size. However, these criteria have been challenged by series of clinical studies and case reports, suggesting prediction AML rupture based on tumor size is not always reliable.The authors searched PubMed using “angiomyolipoma,” “AML,” and “rupture” and reviewed relevant studies. The authors investigated the risk factors of AML rupture using the retrieved literature. The authors also summarized current modalities to evaluate and manage AML.It is established that risk of AML rupture is associated with lesion size. However, genetic abnormality, aneurysm formation, and pregnancy are also risk factors for tumor rupture. Thus, the prediction of AML rupture should be based on a more comprehensive risk assessment system. The management of renal AML and tumor rupture was also discussed in the present paper.The risk of AML rupture is associated with but not exclusive to lesion size. Any decision to intervene AML must be based on multiple factors including risk, symptoms, and auxiliary findings. The majority of patients with TSC (80%) can develop AML, and TSC is caused by mutations in TSC1 (9q34) or TSC2 (16p13) genes. Similar genetic alteration is also observed in patients with lymphangioleiomyomatosis (LAM). Pathologically AML is caused by clonal proliferation of perivascular epithelioid cells predominantly comprised of fat cells, immature smooth muscle cells, and blood vessels; it is currently thought to belong to a family of tumor collectively referred to as perivascular epithelioid cell tumors. According to the histological composition, there are 2 types of renal AML, a classic type and an epithelioid variant. Specially, epithelioid AML is composed of monotypic epithelial cells and possesses malignant and metastatic potential.Angiomyolipoma (AML) is the most common benign mesenchymal neoplasm of the kidney. The incidence of AML in the general population is estimated to be 0.13%, and it is more prevalent in women than in men.,6 The main complication of AML is retroperitoneal hemorrhage caused by tumor rupture, which can be severe and life threatening. Acute bleeding of AML manifest as Lenk's triad, including acute flank pain, abdominal tenderness, and signs of internal bleeding such as hematuria; this urological emergency is also called Wünderlich syndrome.,8 Currently, the risk of tumor rupture is usually determined by tumor size and surgical approach is necessary for tumor size >4 to 8 cm.,10 However, these criteria have been challenged by a series of clinical cases and cohort studies. More importantly, few studies up to date have comprehensively reviewed the risk factors correlated to the rupture and prognosis of AML. Thus, the present paper aimed to review the radiological classification of AML subtypes and provide recent updates of clinical findings associated with AML rupture; by doing so we hope to remind urologists the conventional criteria based on tumor size for AML prognosis may not always be reliable.Most AML patients are usually asymptomatic and the diagnosis of AML is often incidental. The classical triad of symptoms characterized by flank pain, palpable mass, and hematuria appeared in less than half of the individuals.2First, the search term “angiomyolipoma” and “AML” were searched in PubMed and Web of Science, respectively. To avoid ambiguities, MeSH database was used for PubMed search. Then, the “English and Humans” filter was applied to the search results and a total of 1535 articles were found, among which 47 articles significantly related to our topic were included. Additional searches were conducted when any relevant references were discovered in these articles. Finally, 56 articles were reviewed in detail.The ethic approval was not necessary in this study because no human data or animal experiments were involved.3,10 However, a series of clinical studies have reported that hemorrhage and aneurysm formation was not present even in patients with AML >4 cm or with intratumoral aneurysms >5 mm; by contrast, tumor as small as <4 cm may also rupture spontaneously. For example, Gomha et al found that large AML (>10 cm) could also remain stable undergoing conservative management. Prischl and Spottl discovered 9.4% of small AML (<4 cm) could also rupture. More importantly, recent evidences suggest that the rupture of AML may be also influenced by other factors regardless of tumor size.The main complication of AML is retroperitoneal hemorrhage caused by AML rupture, which can be severe and lead to a poor prognosis. Risk factors for AML rupture used to focus on tumor size. It has been commonly proposed that tumor with diameter >4 cm is more likely to develop aneurysm and rupture.Considering these challenges to the traditional criteria as well as many cases of ruptured AML, here we propose that risk of AML rupture should be based on a more comprehensive risk assessment system including tumor size, aneurysm formation, pregnancy, coagulopathy, trauma, hormone level, and comorbidity with TSC/LAM. We believe that the interaction among aneurysm formation and TSC/LAM, pregnancy, and the precipitating factors play central role in AML rupture Fig. . Thus, t3.1TSC1 or TSC2 genes were more likely to suffer from aneurysm, which lead to rapid growth and spontaneous rupture of the tumor.TSC1 or TSC2 mutant genes lead to hyper-activation of mTOR in the regulating pathways in developing fetus, and the disruption of normal angiogenic pathways through hyperactive mTOR signaling may be the mechanism that lead to deranged vascular pathogenesis in the TSC, explaining the tendency of aneurysm formation in TSC individuals.,14 Harrington et al found that a major form of negative feedback inhibition of PI3K resulted from activated growth signaling via mTOR and the p70 S6 kinase (S6K), which is responsible for the development of TSC. In addition, other biochemical mechanisms apart from PI3K, such as PAK2, as well as crosstalk between downstream molecules of mTOR may also aggravate the symptoms of AML and induce hemorrhage.,17Genetic abnormality plays an “initial” role in the complex relationships among all risk factors. It is confirmed that, owing to the uncontrolled mammalian target of rapamycin (mTOR) activation and its angiogenesis effects, TSC patients with loss of  Based on these findings, TSC and LAM significantly increased the risk of aneurysm formation and AML rupture.Because LAM shares the similar genetic characteristics with TSC, patients with LAM were also at an increased risk of AML rupture. By contrast, sporadic AML rarely develop aneurysm or rupture.3.2 Another type of “aneurysm,” pseudoaneurysm, is actually a hematoma restricted by surrounding tissues, and is often caused by traumatic events. Pseudoaneurysms are formed by arteries or arterioles lesion and can cause retroperitoneal hemorrhage and even hypovolemic shock after a secondary rupture.,20Due to the hypervascularity of aneurysms, risk of tumor rupture in AML is related to the presence and size of intratumoral aneurysms, the latter having the strongest association with future risk of rupture. Clinically, image findings described as “aneurysm” are divided into 2 types of pathologic conditions: primary aneurysm and pseudoaneurysm. The primary aneurysms are a “true aneurysm” involving all 3 layers of arterial wall, which can lead to life-threatening retroperitoneal hemorrhage. Apart from aneurysm size, the proportion of angiogenic component in the tumor may also play an important role in rupture. These data are consistent with the findings of Rimon et al in which they further confirmed that large AML with minimal vascularity tend not to bleed.It has been reported that the aneurysm size >5 mm is more strongly correlated with rupture than tumor size > 4 mm. Using an aneurysm size threshold of 5 mm yields a sensitivity of 100% and specificity of 86% for prediction of hemorrhage. By contrast, tumor size threshold of 4 cm for prediction of future hemorrhage presents a lower statistic value with the sensitivity of 100% and specificity of 38%.3.3,25 In particular, spontaneous renal hemorrhage during pregnancy is rare but the consequences may be catastrophic, including maternal shock and intrauterine fetal death. Pregnancy exerts its negative impacts on AML in many ways. First, the increased number of estrogen and progesterone receptors on smooth muscle cells during pregnancy leads to the deceleration of ureter movement; positive receptors for estrogen and progesterone have been found in more than 25% of these cases. Second, the dilated ureter and enlarged uterine may cause slight hydronephrosis. Besides, the increased blood volume and renal plasma flow during the whole term of pregnancy may disturb the hemodynamics and facilitate aneurysm formation. Finally, the increased blood pressure during 24 to 26 weeks of gestation pregnancy may also contribute to aneurysm formation and rupture directly.In the last 5 years, AML in pregnant individuals has aroused increasing concerns. AML during pregnancy is characterized by faster growth, potentially invasive behaviors, and an increased risk of rupture with massive retroperitoneal hemorrhage.Particularly, a series of pathophysiological changes in AML patients during childbirth including increased muscle sensitivity to oxytocin, enhanced abdominal pressure with uterine contraction, and unstable hemodynamics during delivery also lead to tumor rupture easily. Thus, considering the severe complication of AML rupture during pregnancy and childbirth, it has been suggested that women with known AML who intend pregnancy should be treated prophylactically if the tumor is more than 4 cm, even when asymptomatic, to avoid the risk of rupture.3.4 reported a renal AML case with life-threatening rupture and demonstrated that there is a potentially life-threatening association between horse chestnut seed extract-containing products and renal AML. In addition, intracapsular or retroperitoneal hemorrhage of renal AML may result from trauma, even blunt and low-velocity force. In particular, Zengin et al even reported a ruptured renal AML patient caused by hard abdominal physical examination.,29The precipitating factors of tumor rupture are various in clinical practice. Snow et al4AML is usually easy to diagnose with imaging alone. It is very important to make precise diagnosis and clinical evaluation of this disease, considering the serious potential complication of retroperitoneal hemorrhage after tumor rupture. Fortunately, the unique features on ultrasonography (US), computed tomography (CT), and magnetic resonance imaging (MRI) usually make AML easily differentiated from other renal masses. The most crucial aims of image checks are to identify and assess those signs associated with tumor rupture such as aneurysm or pseudoaneurysm formation. Therefore, radiologist and clinicians should always know what image features to expect prior to choosing the imaging techniques. Here, Jinzaki et al summarized and described the imaging features of renal AML  because of inaccurate placement of region of interest measurements and volume averaging of voxels containing renal parenchyma and fat; CT imaging is helpful to definitively characterize large AML, but large AML should be differentiated from other fat-containing retroperitoneal masses, such as liposarcoma. Although there are multiple of CT imaging features for describing AML with minimal fat component, most of these findings are overlapped with renal cell carcinoma; More importantly, although CT evidences including thresholds of 4 cm for tumor size and 5 mm for size of aneurysm have been proposed for prediction of future bleeding, the decision to intervene remains controversial as many AML >4 cm or with intratumoral aneurysms >5 mm do not undergo hemorrhage and remain asymptomatic. Therefore, it has been advised that the decision to treat an asymptomatic patient with AML must be based on multiple factors not exclusive to lesion size. Using this technique, Rimon et al established a scoring system based on CT and DSA appearance to evaluate the hemorrhage risk and to analyze the correlation between hemorrhage and vascularity grading. Their results suggest large AML with minimal vascularity are less likely to bleed, and prophylactic treatment is not necessary.Limitations of CT include relatively poor imaging of small vessels. However, digital subtraction angiography (DSA) can display the vascularity of AML clearly.4.3MRI possesses high sensitivity for detecting fat tissues; thus classical AML can be easily identified by MRI. The T1-weighed imaging of MRI displays fat as hyperintense. Comparing the signal intensity between fat-suppressed and nonfat-suppressed sequences helps to further identify the fatty component. On both frequency selective MRI and chemical shift (CS) MRI, classical AML is present as loss of signal with fat suppression which indicates the presence of fat cells. Fat-poor AML is relatively T2-hyposignal, and epithelioid AML is T2-hyposignal with heterogeneously enhancing or multilocular cysts. However, compared with DSA and Doppler sonography, MRI has limited ability to outline the vascularity of tumor. Consequently, combination of different imaging techniques is necessary for a comprehensive evaluation of AML.It has been established that MRI is useful to differentiate AML from the other renal mass. For example, using the opposed-phase CS-MRI, Israel et al have successfully differentiated renal AML from solid renal masses and hemorrhagic-proteinaceous renal cysts. Their findings suggest “India ink artifact” signal within the renal mass or at the mass–kidney interface is indicative of AML with high sensitivity (100%) and specificity (97.9%).5Management of AML patients must be based on clear diagnosis for AML and risk assessment for tumor rupture. As previously discussed, diagnostic imaging is of definite value. On the other hand, routine check for AML patients, such as hematology, urine test, coagulation, biochemistry, and hormone level, is mandatory, because these factors may predispose patients to higher risk of tumor rupture. In addition, although biopsy for AML possesses unique immunohistochemical staining and is definitive in the diagnosis of AML, it is rarely used for the risk assessment of tumor rupture clinically.Most AMLs do not require treatment because they are benign and asymptomatic. Indications for treatment include suspicion of malignancy, spontaneous hemorrhage causing significant symptoms, risk of rupture, or other complications. Here Jinzaki et al proposed a treatment scheme for AML of different radiologic class Table .5.1; however, repeated bleeding may present if the lesion cannot reach complete morphological recovery. Consequently, repeat yearly or semiyearly CT or US can be performed to evaluate the risk of rupture, and those who choose such conservative management should probably avoid contact activities.Conservation is suitable for asymptomatic lesions <4 cm or some larger but stable masses. Albi et al believed that even Wünderlich syndrome can be also managed conservatively if the hemorrhage is limited and respond to fluid resuscitation5.2 Moreover, this therapy can also reduce the risk of rupture and bleeding.As described in the previous paragraphs, the pathological changes underlying AML rupture is the rapid growth vascularization of the lesion site, and the mTOR plays the central role in the biochemical process of tumor growth and vascularization by acting as a kinase. Recently, mTOR inhibitors (a group of immunosuppressive agents) such as rapamycin and everolimus have been proved to be effective for AML patients by inhibiting vascular epithelial proliferation and reducing the tumor size.TSC1 or TSC2 genes and thus uncontrolled mTOR activation, medication can be especially beneficial to patients with these patients. Therefore, literatures have recommended mTOR inhibitors for TSC/LAM patients to control and reduce tumor size.Because patients with TSC or LAM have genetic abnormalities in 5.3 Moreover, compared with surgical alternatives, embolization possesses several advantages including a low complication rate, less trauma, renal function preserve, and satisfactory short-term (<5 years) outcome.,45 Ewalt et al found that transcatheter embolization of large AML prevented hemorrhage and renal loss; this intervention is minimally invasive and preserves renal function. Wang et al, respectively, reviewed 46 patients who underwent super-selective renal artery embolization (SRAE) for renal hemorrhage, and indicated that SRAE is an effective and minimally invasive method for the control of renal hemorrhage. Choices of embolization agents and methods significantly determine the outcome of embolization and should be guided by the hemodynamics of patients. Development and application of new embolization agents may help interventionist better cope with clinical practice.Controversies over the choice between embolization and surgery mainly focus on the safety, efficacy, renal preserve, and pregnancy. Although both surgery and embolization are useful for patients with AML <4 cm, currently renal artery embolization is recommended as a first-line therapy for bleeding AML and is increasingly used as a preventive treatment for AML at risk of bleeding.,51; Boorjian et al pointed out that embolization had higher risk of relapse and recurrent bleeding compared with partial nephrectomy. Second, it is argued that selective embolization was not devoid of complications; Kara et al recommended RPN as an option for patients with increased risk of rupture.Although embolization is the currently preferred treatment of symptomatic or ruptured renal AML with high safety and well renal preserve, limitations of embolization also exist. First, it should be noted that although embolization induced tumor shrinkage in most patients, tumor shrinkage alone is not a reliable exclusion criterion for recurrent hemorrhage,54; large proportion of vascularity may also require repeated embolization due to the complexity of vascular arrangement; and owing to the radioactive hazard during embolization, it is not applicable to pregnant patients who wish to preserve the fetus.Embolization has limited application in several scenarios: multiple or giant aneurysm, as seen in TSC and LAM respond poorly to embolization; large ratio of fat to vascularity always indicate poor outcome of the procedure5.4 Boorjian et al reviewed patients undergoing NSS from 1970 to 2004 in their institution and concluded NSS offered preservation of renal function and was associated with acceptable complication  and low local recurrence rates. Furthermore, recent development of robotic technology allows surgeons to improve operation outcome by decreasing complication rate and increasing renal preserve. In conclusion, it is believed that surgery will continue to play important roles in the treatment of AML.Surgery was the classical treatment for symptomatic AMLs until the recent emergence of embolization techniques. Nevertheless, surgery still remained an important option for large, symptomatic AMLs. Nephron-sparing surgery (NSS) is now widely accepted as the optimal operation in clinical practice for its feasibility, efficacy, and satisfactory renal preserve. In a retrospective study conducted by Yip et al, they considered that NSS is feasible and effective for renal AML, even for massive AML or after previous rupture, especially when the diagnosis was made by preoperative imaging and/or intraoperative frozen section.5.5As discussed, pregnancy is also a contraindication of embolization. Due to the increased risk of tumor rupture and invasion, termination of pregnancy should be considered in patients diagnosed with AML during early pregnancy.,57,58 Although there are some case reports that multidisciplinary approach may leave the fetus in gestation whilst facilitating radical nephrectomy, management of such cases still remains challenging.It has been advised that women with known AML who intend pregnancy should be treated prophylactically if the tumor is more than 4 cm, even when asymptomatic, to avoid the risk of rupture. The patient will for delivery, gestational week, fetal and mother's state should also be taken into consideration. Surgery is a better choice for full-term pregnancy women with AML as it can manage the renal condition without compromising the safety of mother and fetus; obstetric emergencies, such as fetal distress, dystocia, placenta praevia, are strong indicators for joint surgery of nephrectomy and caesarean delivery. Nephrectomy yielded good prognosis for both mother and fetus in many cases and has proved to be successful in AML during pregnancy.6Renal AML is a common benign tumor composed of dysmorphic blood vessels, smooth muscle, and mature adipose tissue with varying proportions. The main complication of AML is retroperitoneal hemorrhage caused by tumor rupture, which can be severe and life threatening. The clinical intervention of AML is mainly based on classification of the tumor and the risk of AML rupture. It has been widely accepted that AML tumor size >4 cm or intratumoral aneurysms >5 mm is the recommended criteria to determine the risk of tumor rupture. However, these criteria are not always reliable, as many patients with AML >4 cm or with aneurysms >5 mm did not undergo rupture or aneurysms formation. Thus, the current criteria are hardly satisfactory. The authors believe that prediction of AML rupture should be assessed on a more comprehensive risk assessment system including epidemiologic, pathologic features, and imaging evidence. The treatment strategy should be compatible with the risk factors discussed above, because patients with different risk factors showed different tolerance and prognosis. Unfortunately none of the current studies have managed to cover all risk factors. The paucity of literatures available on the prognostic factors of AML also set limit to our review. As more clinical researches and data emerges, it should be expected that a detailed scoring system can be established to help identify patients in need for prophylactic intervention.Formal analysis: Xin Gou.Investigation: Jing Fan.Methodology: Chenyang Wang.Software: Linglong Peng.Writing – original draft: Xinyuan Li.


Ion channels play a crucial role in the development of ischemic brain injury. Recent studies have reported that the blockade of various types of ion channels improves outcomes in experimental stroke models. Amiodarone, one of the most effective drugs for life-threatening arrhythmia, works as a multiple channel blocker and its characteristics cover all four Vaughan-Williams classes. Although it is known that amiodarone indirectly contributes to preventing ischemic stroke by maintaining sinus rhythm in patients with atrial fibrillation, the direct neuroprotective effect of amiodarone has not been clarified. The purpose of this study was to investigate the direct effect of amiodarone on ischemic stroke in mice.Focal cerebral ischemia was induced via distal permanent middle cerebral artery occlusion (MCAO) in adult male mice. The amiodarone pre-treatment group received 50 mg/kg of amiodarone 1 h before MCAO; the amiodarone post-treatment groups received 50 mg/kg of amiodarone immediately after MCAO; the control group received vehicle only. In addition, the sodium channel opener veratrine and selective beta-adrenergic agonist isoprotelenol were used to elucidate the targeted pathway. Heart rate and blood pressure were monitored perioperatively. Infarct volume analysis was conducted 48 h after MCAO. The body asymmetry test and the corner test were used for neurological evaluation.P < 0.05) and improved functional outcomes: lower asymmetric body swing rates  and fewer left turns . In contrast, amiodarone post-treatment did not improve the outcomes after MCAO. The neuroprotective effect of amiodarone pre-treatment was abolished by co-administration of veratrine but not by isoproterenol.Amiodarone pre-treatment and post-treatment reduced the heart rate but did not affect the blood pressure. No mice showed arrhythmia. Compared with the control group, the amiodarone pre-treatment group had smaller infarct volumes (8.9 ± 2.1% hemisphere [mean ± SD] vs. 11.2 ± 1.4%; Amiodarone pre-treatment attenuated ischemic brain injury and improved functional outcomes without affecting heart rhythm and blood pressure. The present results showed that amiodarone pre-treatment has neuroprotective effects, at least in part, via blocking the sodium channels. Intracellular Na+ accumulates and the brain cell membrane becomes depolarized. Excessive glutamate release and subsequent activation of voltage-gated Na+ and Ca2+ channels occur, resulting in neuronal hyperexcitability with further accumulation of intracellular Na+ and Ca2+. As a result, cellular necrosis due to osmotic swelling occurs  × 100% . Total iNeurological function and deterioration were assessed by a blinded observer using the body asymmetry test  and the During the body asymmetry test, animals were suspended by their tails and the direction of head movement was recorded . The triDuring the corner test, a corner was made by attaching two boards (30 cm × 20 cm × 1 cm) at an angle of 30°. A small opening was made along the joint to encourage entry into the corner. Animals were placed midway from the corner. When the animals reached the corner and their vibrissae were stimulated, they reared upward and then turned to either side. The number of left  turns during 10 trials was recorded. The mice with damage to the left cortex are expected to turn more toward the left  side. This test was used to assess the sensorimotor and postural function , 12.P < 0.05 was considered statistically significant.All values are presented as means ± standard deviation (SD). Subgroup comparisons were analyzed using one-way analysis of variance (ANOVA), followed by the Dunnett test. Hemodynamic data were compared among groups by two-way ANOVA for repeated measures. Statistical analysis was performed using Prism 6 software . The sample size of 15 mice per group was sufficient to provide 80% power with an α level of 0.05 to detect a mean difference of 2% in infarct volume.P < 0.05 for all data). No animal demonstrated arrhythmia. There was a trend toward slightly lower systolic blood pressure and diastolic blood pressure in the pre-treatment and the post-treatment groups. However, the difference was not statistically significant  : 0.15–3.1). In contrast, amiodarone post-treatment did not reduce infarct volume  : 0.1–4.0).Representative TTC-stained coronal brain sections are shown in Fig. P < 0.05; 95% CI: 1–31) [Fig. P < 0.05; 95% CI: 0.0–2.4) [Fig. P < 0.05; 95% CI: 0.3–3.4) [Fig. Amiodarone pre-treatment (but not post-treatment) improved functional outcomes of the behavioral tests Figs.  and 4. DIn the present study, we found that amiodarone administered before ischemic brain insult lessened the infarct volume and improved neurological function.+, Ca2+, and K+ channels, Na+-K+-ATPase, and Na+/Ca2+ exchanger. [+ [2+ [+ blockers [+ or Ca2+ channels prevents hyperexcitability and accumulation of Na+ and Ca2+ after ischemic injury by stabilizing the cellular membrane [+ channel blockade in the ischemic brain [ATP channels play a crucial role in pharmacological preconditioning effects against ischemic insult [+ channel can be detrimental. However, amiodarone blocks KATP channels but has no effect on mitochondrial KATP channels [ATP channels creates a neuroprotective effect through the effect on microglia [+-K+-ATPase and Na+/Ca2+ exchanger blockade on the ischemic brain are unclear and controversial [+-K+-ATPase helps to preserve cellular ATP. If the ATP shortage is critical, however, inactivation of Na+-K+-ATPase may result in the further accumulation of cellular Na+ and Ca2+ and exacerbate neuronal death [+/Ca2+ exchanger, an antiporter membrane protein, transports Ca2+ out as Na+ enters the cell [+, Na+/Ca2+ exchanger promotes in the reverse mode, which produces an opposite transporting direction (Ca2+ influx and Na + efflux) [+/Ca2+ exchanger operates depends on the Na+ and Ca2+ transmembrane gradient. These characteristics and the complexity of Na+/Ca2+ exchanger may explain differing results, indicating both neuroprotective and harmful effects of Na+/Ca2+ exchanger blockers on ischemic brain injury [Systemically administered amiodarone passes the blood–brain barrier , 14 and changer. , 16, 17 nger. [+ , Ca2+ [1. [+ [2+ , and K+ blockers , 4. Blocic brain , 4. Amioc insult , 20, blochannels . Interesoversial , 23. Depthe cell . In isch efflux) . The modn injury , 23.2+ influx [Non-electrophysiological effects of amiodarone include inhibitory effects on beta-adrenergic receptors . Blockad+ influx . In addi+ influx –31 and s+ influx –32, whic+ [2+ [+ blockers [We could not identify a neuroprotective effect of amiodarone post-treatment. It is reported that post-treatment with Na+ , Ca2+ [1+ [2+ , and K+ blockers , 4, betablockers , anti-inblockers , 34, andblockers  providesThe clinical significance of our study is that systemically administered amiodarone prior to ischemic brain insult has the potential to improve neurological functional outcomes. Amiodarone is one of the most commonly prescribed drugs for life-threatening and refractory arrhythmias, and it is currently a second-line drug for the treatment of ventricular fibrillation during cardiopulmonary resuscitation . CardiacThis study had several limitations. Since amiodarone has a complex pharmacological profile, there remains a possibility that other mechanisms are involved in the neuroprotective effect of amiodarone observed in this study. Furthermore, amiodarone could have diverse roles and exert different effects based on various factors or the severity of brain injury. Although there was no statistical difference in blood pressure among the groups, there was a trend toward lower blood pressure in the pre-treatment and post-treatment groups. Hypotension is a major side effect of single-bolus amiodarone  that canAmiodarone pre-treatment, but not post-treatment, attenuates ischemic brain injury and improves functional outcomes without affecting heart rhythm and blood pressure. The neuroprotective properties of amiodarone demonstrated in the present study may be explained by its complex pharmacological profile as a multiple ion channel blocker. The results of the present study may indicate that systemically administered amiodarone prior to ischemic brain insult has the potential to improve neurological outcomes. Further studies are needed to elucidate the underlying mechanisms and therapeutic time windows of amiodarone for ischemic brain injury.


Experimentally, two independent methods based on spectrophotometry and mass spectrometry provided evidence that recyclization of the β-lactam ring within the active site of Ldtfm regenerates native nitrocefin. Ring strain is therefore not sufficient to account for irreversible acylation of peptidoglycan transpeptidases observed for most β-lactam antibiotics.β-lactam antibiotics act as suicide substrates of transpeptidases responsible for the last cross-linking step of peptidoglycan synthesis in the bacterial cell wall. Nucleophilic attack of the β-lactam carbonyl by the catalytic residue (Ser or Cys) of transpeptidases results in the opening of the β-lactam ring and in the formation of a stable acyl-enzyme. The acylation reaction is considered as irreversible due to the strain of the β-lactam ring. In contradiction with this widely accepted but poorly demonstrated premise, we show here that the acylation of the L,D-transpeptidase Ldt The latter cell wall polymer is a giant macromolecule of 109 to 1010 daltons that completely surrounds bacterial cells and plays an essential role in counteracting the osmotic pressure of the cytoplasm during the entire cell cycle including cell division2. β-lactams are structure analogues of the peptidoglycan precursors and act as suicide substrates of the transpeptidases3. The enzymes catalyze formation of peptidoglycan cross-links in a two-step reaction4. In the first step, the D,D-transpeptidases interact with an acyl donor containing a pentapeptide stem ending in D-Ala4-D-Ala5, leading to the release of D-Ala5 and to the formation of an ester bond between the carbonyl of D-Ala4 and the catalytic Ser residue of the enzymes. In the second step, nucleophilic attack of the resulting acyl-enzyme by the acyl acceptor generates the peptidoglycan cross-link. Similar to the first step of the transpeptidation reaction, nucleophilic attack of the carbon carbonyl of β-lactams by the catalytic Ser leads to rupture of the amide bond of the β-lactam ring and inactivation of the D,D-transpeptidases. The resulting acyl-enzymes are highly stable, with typical half-lives in the order of several hours, due to low hydrolysis rates. For this reason, the inactivation reaction is considered as irreversible since recovery of enzyme activity through hydrolysis of the drug-enzyme adduct occurs at a timescale that is too large to compromise antibacterial activity.β-lactams remain the corner stone of antibacterial chemotherapy 76 years after the first therapeutic use of an antibiotic, namely penicillin, in 1941. β-lactams kill bacteria by inactivating the D,D-transpeptidases responsible for the last cross-linking step of peptidoglycan synthesis1. PBPs belong to a super family of mechanistically and structurally related active-site serine enzymes that also comprise D,D-carboxypeptidases, endopeptidases, and most β-lactamases responsible for β-lactam resistance by drug detoxification5. The acyl-enzymes formed by the acylation of the Ser residue of β-lactamases are rapidly hydrolyzed leading to turnover and drug detoxification. More recently, the family of β-lactam-interacting enzymes has been enriched by the detection of active-site cysteine L,D-transpeptidases that by-pass the D,D-transpeptidase activity of PBPs in β-lactam-resistant mutants of Enterococcus faecium and Escherichia coli7. These L,D-transpeptidases were also found to be the main peptidoglycan cross-linking enzymes in Clostridium difficile and in mycobacteria8. The L,D-transpeptidases are structurally unrelated to active-site Ser PBPs and cleave the L-Lys3-D-Ala4 or diaminopymelate3-D-Ala4 peptide bond of an acyl donor containing a tetrapeptide stem10. In spite of this difference in substrate specificity, L,D-transpeptidases interact with β-lactams belonging to the carbapenem class11  since these enzymes were routinely identified by covalent labeling with radioactive penicillin followed by gel electrophoresis16. In this study, we have critically evaluated this postulate by studying the acylation of the L,D-transpeptidase Ldtfm from E. faecium by nitrocefin, a chromogenic cephalosporin. We show that this drug-enzyme combination displays high acylation and low hydrolysis rates leading to acyl-enzyme accumulation. This offered the possibility to assay for recyclization of the β-lactam ring by two approaches based on the use of a competing β-lactam or a competing enzyme. We provide evidence that recyclization of the β-lactam ring of nitrocefin in the acyl-enzyme regenerates the native drug that can freely diffuse out of the Ldtfm active site. These data indicate that the irreversible nature of the acylation reaction is not exclusively driven by the strain of the four-membered ring of β-lactams.Due to the strain of the four-membered ring of β-lactams, the acylation reaction is considered as irreversible in the sense that re-sealing of the scissile amide bond of the β-lactam ring is not thought to occur at any significant rate−1 cm−1 . Incubation of Ldtfm (22 µM) with increasing concentrations of nitrocefin (0 to 56 µM) also led to an increase of the absorbance at 486 nm . A minor red shift might occur in the absorbance maximum . Thus, nitrocefin provides a sensitive assay to titrate the active site of Ldtfm.Hydrolysis of the β-lactam ring of nitrocefin (50 µM) by the β-lactamase BlaC (1 µM) in 100 mM sodium phosphate buffer (pH 6.0) produced the expected large increase in the absorbance at 486 nm Fig. . Under t nm Fig. . The inctry Fig. . The spefm (11 µM) by nitrocefin  between native nitrocefin  and nitrocefin in the acyl-enzyme  .Stopped-flow kinetics were performed to assess the efficacy of acylation of Ldtn 1 Fig. , exponenfin Fig. . The slofm (0 to 22 µM) for an extended time period (14 hours) revealed an increase in the absorbance at 486 nm  corresponds to the value of the kinetic parameter k3 since the bulk of Ldtfm is acylated under these conditions  with various concentrations of Ldt nm Fig. , which w nm Fig. . The confm 0 to 2 µM for ons Fig. . The valfm (22 µM) was simultaneously exposed to nitrocefin (50 µM) and imipenem (64 µM) . We therefore concluded that native nitrocefin and nitrocefin bound to the catalytic Cys of Ldtfm in the acyl-enzyme were in equilibrium and that imipenem displaced this equilibrium in favor of the native drug. In the competing acylation step, Ldtfm was initially fully acylated by nitrocefin but imipenem gradually replaced nitrocefin in the enzyme active site since the acylation reaction was irreversible with imipenem but reversible with nitrocefin (Scheme 2). In the control experiment without imipenem, slow hydrolysis of nitrocefin was observed as expected from the kinetics displayed in Fig. LdtµM) Fig. . The kin nm Fig. . This defm with imipenem and nitrocefin, the recyclization reaction generates the native form of Ldtfm, which is expected to be competitively acylated by imipenem and nitrocefin. Recyclization followed by acylation with nitrocefin contributes to a futile cycle with no net impact on the concentration of nitrocefin. This implies that the net rate of nitrocefin recyclization should increase with the concentration of the competing imipenem. Testing recyclization of nitrocefin (50 µM) in the presence of various concentrations of imipenem  revealed the expected increase in the net rate of nitrocefin recyclization  was first incubated with labeled Ldtfm (2.2 nmole) in 100 µl of 5 mM sodium phosphate buffer (pH 6.0). The extent of acylation (ca. 82%) indicated that the bulk of nitrocefin had reacted with Ldtfm  was added and the partition of nitrocefin between the two forms of Ldtfm was determined by mass spectrometry. After 600 s of incubation an equilibrium was reached and 40% of the labeled and unlabeled forms of Ldtfm were acylated by nitrocefin. This result indicates that the β-lactam ring of nitrocefin was resealed within the active site of labeled Ldtfm since this was the only source of native nitrocefin for acylation of unlabeled Ldtfm.Since the reversibility of the acylation reaction was unprecedented for any β-lactam, our next objective was to demonstrate the reversibility of the acylation of Ldttfm Fig. . To brintfm Fig. . Then, uEnterococcus faecium (M512), which was selected in laboratory conditions on media containing increasing concentrations of ampicillin11. Depending upon the growth conditions, the peptidoglycan of the mutant M512 is exclusively cross-linked by the PBPs or by Ldtfm. In the presence of ampicillin (32 µg/ml), the D,D-transpeptidase activity of all PBPs produced by the mutant M512 is inhibited and Ldtfm is the only functional transpeptidase. Antibiotic susceptibility testing in media containing ampicillin (32 µg/ml) was therefore used to assay for inhibition of Ldtfm by nitrocefin. The broth microdilution assay revealed a 64-fold difference between the minimal inhibitory concentration (MIC) of nitrocefin (4 µg/ml) and that of imipenem (0.0625 µg/ml). Thus, nitrocefin was less active than imipenem in spite of a higher rate of acylation of Ldtfm.We have previously reported activation of the L,D-transpeptidase pathway in a mutant of fm by nitrocefin. To the best of our knowledge, this is the first time that reversible acylation is documented for a β-lactam antibiotic. This conclusion is based on two independent approaches. First, we show that the deacylation (recyclization) reaction regenerates the β-lactam ring of nitrocefin based on spectrophotometry, a technique that provided access to kinetic evaluation  but not for others (e.g. imipenem) since simultaneous incubation of Ldtfm with the two drugs led to the rapid formation of the Ldtfm-nitrocefin acyl-enzyme, which was then slowly replaced by the Ldtfm-imipenem acyl-enzyme. The basis for this difference remains unknown, but it is tempting to speculate that it involves the fate of the negative charge that develops on the β-lactam nitrogen upon rupture of the β-lactam ring   and cephalotin (8.3 ± 3.3 M−1 s−1) or penams such as ampicillin (8.7 ± 0.3 M−1 s−1)14. Since cephalotin and nitrocefin are two cephalosporins harboring the same side-chain at positionC7 , with a lesser impact originating from differences in their side-chains21. These observations suggest that the high efficacy of acylation of Ldtfm by nitrocefin depends upon an effect of the side-chain on the reactivity of the β-lactam ring, as opposed to a direct interaction of the side-chain with the enzyme. This effect of the side-chain may involve the polarization of the carbon-nitrogen bond of the β-lactam ring of nitrocefin.The efficacy of acylation of Ldt−1) Fig.  was high23. For most cephalosporins, irreversibility could result from the loss of a leaving group in the C3 side-chain  of nitrocefin  were prepared in 100% DMSO and stored at −20 °C. Dilutions were extemporaneously prepared in water. Stock solutions of imipenem (provided by Merck) were prepared extemporaneously in water.fm (residues 341 to 466) and of a soluble fragment of the β-lactamase BlaC from M. tuberculosis (residues 39 to 306) have been previously described24. BlaC and Ldtfm were produced in E. coli BL21(DE3) cells grown in brain heart infusion broth (BHI)24. Labeling of Ldtfm was obtained in M9 minimal medium containing 13C glucose and 15N NH4Cl14. Ldtfm and BlaC were purified from clarified lysates by affinity and size-exclusion chromatography in 100 mM sodium phosphate buffer (pH 6.4) and in 25 mM Tris-HCl (pH 7.5) containing 300 mM NaCl, respectively25. The purified enzymes were concentrated by ultrafiltration  and stored at −65 °C in the same buffers.The plasmid constructs for production of the catalytic domain of LdtAll kinetics were performed at 20 °C in 100 mM sodium phosphate (pH 6.0) either in a spectrophotometer  coupled to a stopped-flow apparatus  or in a spectrophotometer (Cary 300) equipped with an automatic sample changer (Varian SA).fm-β-lactam adducts was tested by incubating Ldtfm with β-lactams at 20 °C in 5 mM sodium phosphate buffer (pH 6.0). Five µl of acetonitrile and 1 µl of 1% formic acid were extemporaneously added, and the reaction mixture was directly injected into the mass spectrometer  at a flow rate of 0.05 ml/min . Spectra were acquired in the positive mode, as previously described11.The formation of Ldt5 cfu/ml. Plates were incubated for 24 h at 37 °C and the minimal inhibitory concentration was defined as the minimal concentration that prevented visible growth.The microdilution assay was performed in BHI broth containing 32 µg/ml of ampicillin (Euromedex) in 96-well plates. Each well (200 µl) was inoculated with overnight cultures to obtain 5 × 10Supporting Information


Mentoring continues to build momentum among startups and established enterprises due to its positive impact on individuals and organizations. Unlike previous studies, this research focuses on mentoring higher level leadership, such as the CEO, and demonstrates its unique relationship to organizational innovativeness. Our sample included 200 mentored executives and entrepreneurs who personally identify and exploit opportunities. Our findings confirm that mentoring top leaders positively relates to their perceived innovativeness of the organization and that the relationship is mediated by these leaders’ perception of psychological safety within the organization. Our findings also confirm that the relationship is negatively moderated by these leaders’ cognitive adaptability. The reliability and validity of the results have been proved by using confirmatory factor analysis and advanced regression analytics. As a result, this work demonstrates the value of mentoring top leadership and advocates the importance of establishing a psychologically safe environment to inspire not only top leadership to try new avenues but also for all those within the organization to speak up and speak out. Additionally, our findings encourage organizations to proactively and selectively prioritize mentoring among top leadership, taking into account their differing levels of cognitive adaptability. Finally, further research could focus on how to provide greater support for mentors of higher level leaders. Whether organizations are new or well established, they must be innovative and able to change in order to survive and thrive . HoweverThe relationship between mentoring top leaders and their perception of organizational innovativeness warrants further research for at least three reasons. First, mentoring and innovating share common ground. Both are a form of social interaction and contribute to the process of learning something new . HoweverAnother important area of study is the intermediary process of the mentor influencing the mentee because this process facilitates mentoring outcomes . For exaFinally, in spite of the accumulated research on the various effects of moderation on mentoring , there iIn summary, this study examines the value of mentoring top leadership and the conditions under which its relationship with perceived organizational innovativeness can be mediated by psychological safety and moderated by cognitive adaptability.Mentoring is a socially based learning process between mentor and mentee; since there are over 50 definitions in circulation , it is dMentoring is significant in the development of innovativeness for several reasons. First, Hypothesis 1: The quality of mentoring top leaders will relate to their perception of organizational innovativeness.This study defines psychological safety as an individual’s belief to be safe without fear of negative results . As explThird, psychological safety relates to innovativeness because in order to innovate, one must try new things. Trying new things implies some form of risk taking. Consequently, the safer individuals feel the more likely they are to explore and discover . Fourth,Hypothesis 2: The mentees’ perception of psychological safety in the organization mediates the relationship between the quality of mentoring they receive and their perception of organizational innovativeness.In order to keep up with today’s unprecedented rate of innovation, some scholars have suggested that “successful future strategists will exploit an entrepreneurial mindset [which is] the ability to rapidly sense, act, and mobilize, even under uncertain conditions” . ImpliedOriginally, in the field of entrepreneurship, cognition research was used to better understand opportunity recognition . In a prSuccessful executives or entrepreneurs (and therefore their companies) are swift to modify and adjust depending on what they discover, rather than stubbornly persisting regardless of the feedback or data they receive. Obviously, entrepreneurs and executives need to be persistent but the principle of persistence can often be misapplied when pursing a less innovative path for the sake of not quitting. Top leaders need to develop the ability to recognize new information and then change the way they view the world in order to create something different that has value. Cognitive adaptability can help entrepreneurs and executives alike to do just that: become more innovative, and thus lead their organizations to do and become likewise .We have chosen to study cognitive adaptability as a moderator between the quality of mentoring top leaders and their perception of organizational innovativeness for at least three reasons. First, cognitive adaptability is linked to mentoring because mentoring fosters the kind of self-reflection and metacognition that results in cognitive adaptability . Second,We predict that effectively mentoring top leaders has its greatest impact on their perceived organizational innovativeness when cognitive adaptability is low. Top leaders already high in cognitive adaptability  gain less through quality mentoring because of redundancies in both what the mentoring functions do and what the mentee naturally does; thus, affecting their perception of innovativeness at a less dramatic rate. Likewise, when top leaders with lower levels of cognitive ability are effectively mentored, perceived organizational innovativeness increases at a greater rate than in those with a higher level of cognitive ability. Indeed, it is likely that top leaders low in cognitive adaptability would be less willing to take advice from a mentor because they are not as flexible, thereby not changing behavior. However, all leaders in this study have received mentoring, which suggests that they at least had a desire to move forward in some way and may not be as rigid as that argument implies. Those with low cognitive adaptability are possibly just less experienced or less natural in that ability. Thus, we suggest that if high levels of the mentoring functions mentioned previously occur . The average length of tenure in the organization’s top leadership position was 5.35 years (SD = 6.44). The participants’ organizations had been established for an average of 20.2 years (SD = 28.15). Approximately 39 percent of the organizations had over 100 employees and the organizations represented approximately 45 different industries such as healthcare, textiles, real estate, and education. The mentors of these executives were typically members of the board of directors or were outside experts.The screening and data collection took place over 6 months, after which approximately 303 candidates qualified for the study. Only 200 participated by taking our questionnaire (a 66 percent response rate). Because of the global reach of these executive networks, the participants came from 14 countries; 43 percent from Western countries and 57 percent from Asia. The countries include Canada, China, Cuba, France, Hong Kong, Japan, Korea, New Zealand, The Netherlands, Singapore, Switzerland, Taiwan, The United Kingdom, and The United States. Women represent 18.5 percent of the sample. The mean age of participants was 37.98 , indicating that these dimensions were distinctive and the overall construct was collectively reflective. The Cronbach’s alpha was 0.82.The items were adapted from prior research so they could be worded in context with the goals of our study. Three items were used to measure mentoring’s vocational support function from It was measured using a five-point scale from It was measured using an eight-item scale developed by It was measured using a shortened version of the cognitive adaptability scale from α’s for the 10- and 35-item scales were 0.71 and 0.86, respectively. We also tested the relationship between cognitive adaptability and individual innovative behavior to assess the comparative criterion-related validity, comparing the 10- and the 35-item scales. The 10-item scale was significantly related to individual innovative behavior  as well as the full 35-item scale .Given that we did not use the full version of the scale, we assessed the validity of the shortened scale by comparing these two versions of the scale using an independent field sample where participants evaluated their cognitive adaptability by using the full version of the scale. We obtained a sample of 183 MBA students (57 percent response rate) who rated themselves using the 35-item cognitive adaptability scale. We also asked them to evaluate their individual innovative behavior using We ran a confirmatory factor analysis at first to test the model fit by AMOS 22.0, adopting five widely reported and recommended indices . The medEven though our study was not in the area of medical research and the data in our study was voluntarily self-reported, we followed applicable research procedures in accordance with the Helsinki Declaration as revised in 2013. Our research was approved by Zhejiang University’s Global Entrepreneurship Research Center Committee. All participants in our study were provided sufficient information to be able to give an informed consent to take part in this study. Research respondents were ensured confidentiality and anonymity. All participation was voluntary. We confirm this research is independent and impartial.Table 1 presents summary statistics and bivariate correlations among the variables.2  = 416.54, CFI = 0.92, TLI = 0.91, RMSEA = 0.07, and SRMR = 0.06.We ran a confirmatory factor analysis to test whether our hypothesized model captured distinct constructs. The results show that the hypothesized 4-factor model fits the data acceptably and the hypothesized model captures distinct constructs, with χTable 2 presents the results of a regression analysis of the mediating effect . As shown in Table 2, the total effect of the quality of mentoring on organizational innovativeness is significantly positive , thus supporting Hypothesis 1. Table 2 also presents the direct effect of the quality of mentoring on organizational innovativeness. We also found that the model fit of this mediating effect is acceptable .We adopted bootstrap methods to test the mediating effect by SPSS PROCESS macro (version 2.15), which is concerned with indirect effect . We alsoTable 3. The conditional effect varies at different levels of cognitive adaptability . Figure 2 displays the interactive effects of the quality of mentoring and cognitive adaptability on perceived organization innovativeness, which shows that when top leaders with lower levels of cognitive ability are effectively mentored, perceived organizational innovativeness increases at a greater rate from mentoring than those with a higher level of cognitive ability.The moderating effect was also tested by SPSS PROCESS macro (version 2.15). The coefficient of XW on Y was –0.13 , showing that cognitive adaptability negatively moderates the effect of mentoring on innovativeness. Therefore, Hypothesis 3 is supported. The conditional effect of mentoring on innovativeness was computed by PROCESS, as shown in This study confirms that the quality of mentoring top leaders does positively relate to their perception of organizational innovativeness and that the relationship is mediated by their perception of psychological safety within the organization and negatively moderated by their cognitive adaptability. These outcomes offer theoretical and managerial implications. Limitations are also discussed.Recent empirical studies  considerIn addition, this study responds to the call for more research on the mediating influence of psychological safety in mentoring  and giveThe findings from this research have practical implications as well. Innovation is “the source of sustained advantage for most companies” and depends upon the individual expertise of those who lead the organization . In thatThis study also helps spotlight the vital role psychological safety plays within the organization. According to social learning theory, people are social creatures and learn by observation and repeat what they learn . As top The results regarding the negative moderating role of cognitive adaptability suggest that organizations need to be able to identify those leaders who have the most need of mentoring, in other words those who are less cognitively adaptable, either through inexperience or because of natural tendencies. Valuable resources are usually limited and so it requires a careful assessment of where appropriate executive leadership mentors should be assigned. Additionally, those executives or entrepreneurs who choose their own mentor, should choose one who is an expert and different enough from themselves in order to assist top leaders to become more dynamic, self-aware, strategic, and flexible.Finally, in a business environment where the only thing constant is change, individuals and organizations need to learn how to embrace change and transform their culture in order to innovate at today’s unprecedented rate. Given individuals naturally tend to resist change in organizations because it can be a painful process , this stNotwithstanding this study’s implications and contributions, it has a number of limitations. First, this study was limited in testing the causal inferences between variables by using a cross-sectional design. A longitudinal design would be more ideal for examining how mentoring top leadership works within different time periods. Second, the data is self-reported, which brings with it several common criticisms. However, although the data is self-reported, using top leadership ratings as the measures has advantages because such ratings are usually based on a broader perspective that comes with the higher level position and arguably commands the most weight in terms of judgment. Third, it can be debated that in many circumstances, whether or not an organization is innovative depends upon situational dynamics outside the CEO or entrepreneur’s control, which an individual mentor is also not likely to be able to change or alter. However, although organization innovativeness can be influenced by factors beyond the leadership’s control, it can also be said that CEOs or entrepreneurs from the top down have the ability to impact organizational innovativeness.In conclusion, this study reveals three main findings. First, the quality of mentoring top leaders is linked to their perception of organizational innovativeness, which means effective mentoring is one potential way to increase innovativeness. Second, top leaders’ perception of psychological safety within the organization mediates the relationship between the quality of mentoring and their perception of organizational innovativeness, which means effective mentoring is another way to improve innovativeness via the development of psychological safety. Third, cognitive adaptability negatively moderates the relationship between mentoring and innovativeness, which means effective mentoring can potentially help leaders with lower levels of cognitive adaptability foster innovativeness within the organization. As a result, this study demonstrates the value of mentoring top leadership. Furthermore, this study advocates that it is important to establish a psychologically safe environment to inspire not only top leadership to try new avenues but also to encourage/enable all those within the organization to feel free to express their opinions and ideas. Additionally, our findings encourage organizations to proactively and selectively prioritize mentoring among top leadership taking into consideration their differing levels of cognitive adaptability. Future studies should examine the differences between higher and lower level mentoring and the strategies that cause mentoring top leadership to be effective. Finally, further research could focus on how to provide greater support to the mentors of top leaders.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Furthermore, we found that CRAC/Orai channels may represent the molecular candidate of SOC in intestinal epithelium. CCh increased intracellular Ca2+ but not cAMP, and Ca2+ signaling mediated duodenal Cl- and HCO3- secretion in wild type mice but not in CFTR knockout mice. CCh induced duodenal ion secretion and stimulated PI3K/Akt activity in duodenal epithelium, all of which were inhibited by selective PI3K inhibitors with different structures. CCh-induced Ca2+ signaling also stimulated the phosphorylation of CFTR proteins and their trafficking to the plasma membrane of duodenal epithelial cells, which were inhibited again by selective PI3K inhibitors.Activation of muscarinic receptors by carbachol(CCh) stimulated mouse duodenal 2+ imaging was performed on HT-29 cells.Functional, biochemical and morphological experiments were performed to examine ion secretion, PI3K/Akt and CFTR activity of mouse duodenal epithelium. Ca2+ signaling plays a critical role in intestinal ion secretion via CRAC/Orai-mediated SOCE mechanism on the serosal side of epithelium. We also demonstrated the molecular mechanisms of Ca2+ signaling in CFTR-mediated secretion via novel PI3K/Akt pathway. Our findings suggest new perspectives for drug targets to protect the upper GI tract and control liquid homeostasis in the small intestine.Ca Epithelial ion transports are under control of several neuro-humoral factors, including ACh, 5-HT, PGs, nitric oxide (NO), and capsaicin-sensitive afferent neurons cyt homeostasis in native epithelial cells of the small intestine are still unclear.Moreover, although it is known that Caderstood . It is gr medium . Classic+ stores , 6. Thesphocytes , 8, whicnel Orai , 10. Hownel Orai  and colonel Orai , 13. So - and of HCO3- secretion in the small intestine is on cAMP- and cGMP-dependent regulatory pathways, which mediates several membrane ion channels to contribute to epithelial ion transports. The cystic fibrosis transmembrane conductance regulator (CFTR) is one of the critical channels in the luminal membrane of enterocytes cyt per se is enough to trigger duodenal transepithelial secretion through a novel PI3K/AKT/CFTR pathway. This study not only reveals that Ca2+ signaling is critical to activate CFTR-mediated epithelial ion transportsin the small intestine, but also provides novel insights into the detailed mechanisms of Ca2+-dependent transepithelial Cl- and HCO3- secretion.The duodenal mucosa, due to its strategic location between the stomach and other segments of the small intestine, senses luminal nutrients and regulates duodenal epithelial ion transports, particularly Clric acid . Therefo2+. CCh (100 μM) markedly increased duodenal short-circuit current (Isc), leading to intracellular Ca2+ release via IP3 receptors on the endoplasmic reticulum (ER) membrane cyt by digital Ca2+ imaging. CCh(100 μM) immediately induced Ca2+ signaling in HT-29 cells  first caused a rapid increase in [Ca2+]cyt due to intracellular Ca2+ release from the ER to the cytosol . After Ca2+ release from the ER was complete, restoration of extracellular Ca2+ (2Ca) caused an additional increase in [Ca2+]cyt due to SOCE mechanism . As shown in Figure We further examined the role of CRAC/Orai channels in SOCE mechanism. In HT-29 cells superfused with Cal Figure , left. Am Figure , right. 2+ signaling could stimulate duodenal Isc through Ca2+-activated Cl- channels (CaCC), we applied commonly used CaCC blocker niflumic acid (NFA). At the concentrations of 100-300 μM, it did not affect CCh-induced duodenal Isc , a selective potent CaCC blocker did not affect CCh-induced duodenal Isc either , a highly potent and specific CFTR inhibitor cyt acts as an universal second messenger to regulate many different cellular functions in a variety of cells [2+]cyt is considered as an important regulator of intestinal ion transports cyt signaling are pleiotropic primary second messengers that regulate all secretory epithelia functions. Since cAMP pathway has been extensively studied, it is questioned if pure Ca2+ signaling is critical for intestinal ion secretion. Moreover, mutual regulation of cAMP and Ca2+ signaling is referred to as crosstalk, while integration of their effects can result in an additive or synergistic physiological response cyt).The HT-29 cell lines used in this work were obtained from the American Type Culture Collection  and were cultured at 37°C under 5% CO-/-) mice and their wild-type littermates (CFTR+/+), which were established as described previously orthophosphate from Amer-sham; Anti-PI3K p85 was obtained from Upstate Biotechnology; Anti-phospho-Akt S473 antibody and anti-Akt antibody were obtained from Cell Signaling; All other chemicals in solutions were obtained from Sigma and Calbiochem.The following drugs were used: carbachol, Forskolin, wortmannin, LY294002, and rapamycin from Sigma; CFTRinh-172 from Calbiochem; [


The purpose of this study was to determine whether methanol and its metabolite, formic acid, affect Ca2+ entry into and aggregation of human platelets were measured by spectrofluorometry using Fura-2/AM as an indicator and the light transmission method, respectively.Ca2+ entry into platelets induced by thrombin, thapsigargin, or OAG. Platelet aggregation induced by thrombin, thapsigargin, or OAG was significantly inhibited by formic acid at toxic concentrations (0.01% or higher). Ca2+ entry into platelets induced by thrombin or thapsigargin was also significantly inhibited by formic acid at 0.01% or higher, while that induced by OAG was not affected by formic acid at 0.005 and 0.01% and augmented by that at 0.02%.Thrombin-induced platelet aggregation was significantly augmented by methanol at pharmacological concentrations (0.5–2%) in a concentration-dependent manner. Methanol at 2% significantly attenuated thapsigargin-induced platelet aggregation, which was not significantly affected by lower concentrations (0.5 and 1%) of methanol. Methanol (0.5–2%) did not significantly affect platelet aggregation induced by 1-oleoyl-2-acetyl-sn-glycerol (OAG), or Ca2+ entry into platelets. Formic acid at toxic concentrations has an inhibitory action on platelets aggregation, which was partly explained by the reduction of Ca2+ entry into platelets.Methanol at pharmacological doses has diverse effects on platelet aggregation, depending on the aggregation stimuli, without affecting Ca Alcohol drinking is known to have diverse effects on the risk of cardiovascular disease that depend on the amount of ethanol consumed; the risk is reduced by moderate drinking but is increased by excessive drinking , 2. The Methanol, another variant of alcohol, is used often in industry and is known to cause optic nerve injury and metabolic acidosis through the production of formic acid in vivo . However2+ entry is a crucial step in the platelet activation process i was ascertained using the fluorescent Ca2+ indicator Fura-2. Fura-2 is a ratiometric fluorescent indicator dye that binds to free intracellular calcium. Fura-2-acetoxymethyl ester (Fura-2/AM) is a membrane-permeant derivative of Fura-2 and is often used for measuring intracellular calcium. The washed platelets were loaded with Fura-2/AM  at 37 °C for 30 min. After loading, the platelets were washed once with Ca2+- and Mg2+-free Tyrode solution buffered by Hepes  (pH 7.4) containing 1 mM EGTA, and they were resuspended in 2 ml of a Ca2+-free Tyrode solution buffered by Hepes  (pH 7.4) .[Ca2+]i were determined using a ratio (R) of fluorescence intensity (F) of F340/F380. The fluorescence after sequential addition of 0.25% Triton X-100 and EGTA (5 mM) to the platelet suspension provided the maximum fluorescence ratio (Rmax) and minimum fluorescence ratio (Rmin), respectively. [Ca2+]i was calculated using the following formula i calculated by subtraction of the basal [Ca2+]i level from the maximum [Ca2+]i level after stimulation.Fluorescence measurements were carried out with a dual-wavelength spectrofluorimeter  using a 0.4-ml cuvette maintained at 37 °C. The wavelengths used for excitation were 340 and 380 nm, and the wavelength used for emission was 510 nm. The fractional changes in i measurement except for the volume of the cuvettes.Platelet aggregation was measured using platelets suspended in Ca2+-free medium in the cuvette for 3 min and then stimulated with each stimulant . At 1 min after the addition of each stimulant, the platelets were pretreated with methanol or formic acid at each concentration or a vehicle (distilled water) for 1 min. Then, Ca2+ entry and aggregation were induced by adding CaCl2 (0.5 mM) to the cuvette. It is known that threshold limit value (TLV) for methanol in the industry is 200 ppm, and exposure to this concentration of methanol causes blood methanol concentration of about 10 mg/L (0.00126%) [Platelets were stabilized in a nominally Camg/L 0.006% [12]. .00126%)  and 1.75.00126%) , which c.00126%)  or 16.9 Methanol  and formic acid  purchased from Wako Pure Chemical Co., Osaka, Japan, were prepared at the time of use and diluted with distilled water. Thapsigargin , OAG (Sigma), and Fura-2/AM  were dissolved in dimethylsulfoxide to make stock solutions of 1, 100, and 5 mM, respectively, and stored at − 80 °C. Bovine thrombin  was dissolved in distilled water to make a stock solution of 1 U/μl and was stored at − 80 °C.F test. p values less than 0.05 were regarded as significant.The data are presented as means ± standard deviations. Statistical analysis was performed using the analysis of variance (ANOVA) followed by Scheffé 2+ entry into platelets (b) induced by thrombin. Methanol at these environmental and toxic concentrations significantly affected neither platelet aggregation nor Ca2+ entry into platelets. Platelet aggregation and Ca2+ entry into platelets induced by thapsigargin and OAG were also not significantly affected by methanol at 0.001, 0.02, and 0.2% (data not shown).Figure Figure 2+ entry into platelets corresponding to their aggregation induced by thrombin (a), thapsigargin (b), and OAG (c). Methanol at the above concentrations did not significantly affect Ca2+ entry into platelets induced by any of the three stimulants.Figure Figure 2+ entry into platelets corresponding to their aggregation induced by thrombin (a), thapsigargin (b), and OAG (c). Formic acid at 0.01 and 0.02% significantly inhibited Ca2+ entry into platelets induced by thrombin and thapsigargin. Formic acid at 0.005% also significantly reduced Ca2+ entry into platelets induced by thapsigargin. On the other hand, formic acid at a higher concentration of 0.02% but not at 0.005 and 0.01% significantly augmented Ca2+ entry into platelets induced by OAG.Figure p < 0.05), 7.017 ± 0.013 (p < 0.01), and 6.234 ± 0.085 (p < 0.01), respectively.Additions of formic acid at 0.005, 0.01, and 0.02% to the solution suspending platelets lowered its pH (means ± standard deviations) from 7.424 ± 0.005 to 7.299 ± 0.043 , while thapsigargin-induced aggregation was inhibited by a higher concentration (2%) of methanol but was not significantly changed by lower concentrations. Platelet aggregation is known to depend strongly on Ca2+ entry [2+ entry pathways would be affected by methanol. In this study, we used three platelet activating stimulants that act through different mechanisms. Thrombin activates its receptors, resulting in hydrolysis of phosphoinositides and subsequent production of two signal transduction messengers, inositol trisphosphate and diacylglycerol. The former induces mobilization of Ca2+ from its intracellular stores, which triggers transmembranous Ca2+ entry, so-called capacitative Ca2+ entry, through TRPC  1/4/5 channels [2+ entry [2+ entry induced by any of the stimulants used in this study. Therefore, the effects of methanol on platelet aggregation are thought to be independent of Ca2+ entry, and methanol is suggested to affect signaling pathways in platelets that are downstream of the increase in intracellular Ca2+ concentrations. Further studies are needed to clarify the mechanisms for the diverse effects of methanol on platelet aggregation induced by thrombin and thapsigargin.Although environmental and toxic concentrations of methanol did not affect platelet aggregation and Ca2+ entry , and thuchannels . The lat2+ entry , 19. ThaBlood concentrations of methanol in its acute intoxication have been reported to be about 0.2% , 14. Thu2+ entry in platelets in response to thrombin and thapsigargin. Therefore, formic acid inhibits platelet aggregation through attenuating capacitative Ca2+ entry. Since thrombin is a physiological agonist for platelet aggregation, there is a possibility that bleeding tendency due to attenuation of platelet aggregation occurs in patients with methanol poisoning, and further clinical studies are needed to test this hypothesis. Platelet aggregation induced by OAG was also attenuated in the presence of formic acid, while corresponding Ca2+ entry in platelets induced by OAG was not affected by formic acid at lower concentrations (0.005 and 0.01%) and was rather augmented by formic acid at a higher concentration (0.02%). Thus, in addition to the inhibitory action on capacitative Ca2+ entry, there may be another inhibitory mechanism, besides action on Ca2+ entry, for formic acid-induced inhibition of platelet aggregation. This is the first study showing the inhibitory action of formic acid on Ca2+ entry into platelets and subsequent aggregation of them. Further studies are also needed to clarify the Ca2+ entry-independent pathway for platelet aggregation that is inhibited by formic acid.On the other hand, formic acid, the final metabolite of methanol in vivo, at toxic concentrations showed inhibitory effects on platelet aggregation and corresponding Ca2+ entry into and subsequent aggregation of platelets were decreased under the condition of extracellular acidosis (pH 6.9) and were increased under the condition of extracellular alkalosis (pH 7.9) [2+ entry and subsequent aggregation by extracellular acidosis (pH 6.9) [2+ entry and subsequent aggregation resulted mainly from extracellular acidosis induced by formic acid. This explanation is plausible because metabolic acidosis occurs in patients with severe methanol poisoning [The pH of the solution suspending platelets was lowered by the addition of formic acid 0.005–0.02%) in a concentration-dependent manner. We previously reported that thapsigargin-induced Ca(pH 7.9) . Thus, p(pH 6.9)  were com–0.02% inoisoning .2+ entry and subsequent platelet aggregation. In our previous study, ethanol at its toxic concentrations (0.5–2%) inhibited platelet aggregation in a concentration-dependent manner irrespective of platelet stimuli [2+ entry were complicated. Ethanol decreased Ca2+ entry induced by thapsigargin and increased that induced by OAG and did not significantly affect that induced by thrombin [2+ entry mediated by TRPC1/4/5 and TRPC3/6/7 channels, and these actions were thought to be canceled when platelets were stimulated with thrombin, which activates both TRPC1/4/5 and TRPC3/6/7 channels. In addition to these actions on Ca2+ entry, ethanol has an inhibitory effect on platelet aggregation independent of Ca2+ entry. Thus, the effects of ethanol on Ca2+ entry into and aggregation of platelets are quite different from the effects of methanol reported in the present study. Figure 2+ channels in the plasmalemma but also various intracellular organelles. Further studies are needed to determine the reasons for the differing actions of methanol and ethanol on Ca2+ entry into and aggregation of platelets, and to clarify how these actions are related to the differences in the structures of these alcohol variants.Table  stimuli . The effthrombin . Thus, e2+ entry in platelets. Thus, methanol has diverse effects on platelet aggregation, depending on the aggregation stimuli, without affecting Ca2+ entry into platelets. Formic acid at toxic doses (0.01% or higher) showed significant inhibitory actions on platelet aggregation and corresponding Ca2+ entry in platelets, which may be caused by extracellular acidosis. Therefore, it is suggested that, in methanol intoxication, platelet aggregation is not affected by methanol itself but is inhibited by its metabolite, formic acid, resulting in bleeding tendency, which may be involved in etiology of complicated brain hemorrhage [Methanol, at pharmacological doses (0.5% or higher) but not at toxic doses, had diverse actions on platelet aggregation—levels of aggregation induced by thrombin and thapsigargin were increased and decreased, respectively, by methanol. On the other hand, methanol showed no significant effects on Camorrhage .