Cognitive Training for Reduction of Delirium in Patients Undergoing Cardiac Surgery: A Randomized Clinical Trial.

IMPORTANCE: Postoperative delirium is a common and impactful neuropsychiatric complication in patients undergoing coronary artery bypass grafting surgery. Cognitive training may enhance cognitive reserve, thereby reducing postoperative delirium. OBJECTIVE: To determine whether preoperative cognitive training reduces the incidence of delirium in patients undergoing coronary artery bypass grafting. DESIGN, SETTING, and PARTICIPANTS: This prospective, single-blind, randomized clinical trial was conducted at 3 university teaching hospitals in southeastern China with enrollment between April 2022 and May 2023. Eligible participants included those scheduled for elective coronary artery bypass grafting who consented and enrolled at least 10 days before surgery. INTERVENTIONS: Participating patients were randomly assigned 1:1, stratified by site, to either routine care or cognitive training, which included substantial practice with online tasks designed to enhance cognitive functions including memory, imagination, reasoning, reaction time, attention, and processing speed. MAIN OUTCOMES AND MEASURES: The primary outcome was occurrence of delirium during postoperative days 1 to 7 or until hospital discharge, diagnosed using the Confusion Assessment Method or the Confusion Assessment Method for Intensive Care Units. Secondary outcomes were postoperative cognitive dysfunction, delirium characteristics, and all-cause mortality within 30 days following the operation. RESULTS: A total of 218 patients were randomized and 208 (median [IQR] age, 66 [58-70] years; 64 female [30.8%] and 144 male [69.2%]) were included in final analysis, with 102 randomized to cognitive training and 106 randomized to routine care. Of all participants, 95 (45.7%) had only a primary school education and 54 (26.0%) had finished high school. In the cognitive training group, 28 participants (27.5%) developed delirium compared with 46 participants (43.4%) randomized to routine care. Those receiving cognitive training were 57% less likely to develop delirium compared with those receiving routine care (adjusted odds ratio [aOR] 0.43; 95% CI, 0.23-0.77; P = .007). Significant differences were observed in the incidence of severe delirium (aOR, 0.46; 95% CI, 0.25-0.82; P = .01), median (IQR) duration of delirium (0 [0-1] days for cognitive training vs 0 [0-2] days for routine care; P = .008), and median (IQR) number of delirium-positive days (0 [0-1] days for cognitive training vs 0 [0-2] days for routine care; P = .007). No other secondary outcomes differed significantly. CONCLUSIONS AND RELEVANCE: In this randomized trial of 208 patients undergoing coronary artery bypass grafting, preoperative cognitive training reduced the incidence of postoperative delirium. However, our primary analysis was based on fewer than 75 events and should therefore be considered exploratory and a basis for future larger trials. Trial Registration: Chinese Clinical Trial Registry Identifier: ChiCTR2200058243.

Predictions of Aeroengines' Infrared Radiation Characteristics Based on HKELM Optimized by the Improved Dung Beetle Optimizer.

To solve the problems of high computational cost and the long time required by the simulation and calculation of aeroengines' exhaust systems, a method of predicting the characteristics of infrared radiation based on the hybrid kernel extreme learning machine (HKELM) optimized by the improved dung beetle optimizer (IDBO) was proposed. Firstly, the Levy flight strategy and variable spiral strategy were introduced to improve the optimization performance of the dung beetle optimizer (DBO) algorithm. Secondly, the superiority of IDBO algorithm was verified by using 23 benchmark functions. In addition, the Wilcoxon signed-rank test was applied to evaluate the experimental results, which proved the superiority of the IDBO algorithm over other current prominent metaheuristic algorithms. Finally, the hyperparameters of HKELM were optimized by the IDBO algorithm, and the IDBO-HKELM model was applied to the prediction of characteristics of infrared radiation of a typical axisymmetric nozzle. The results showed that the RMSE and MAE of the IDBO-HKELM model were 20.64 and 8.83, respectively, which verified the high accuracy and feasibility of the proposed method for predictions of aeroengines' infrared radiation characteristics.

Comparison of Carotid Blood Flow Measured by Ultrasound and Cardiac Output in Patients Undergoing Cardiac Surgery.

BACKGROUND: In general, cerebral blood flow accounts for 10-15% of cardiac output (CO), of which about 75% is delivered through the carotid arteries. Hence, if carotid blood flow (CBF) is constantly proportional to CO with high reproducibility and reliability, it would be of great value to measure CBF as an alternative to CO. The aim of this study was to investigate the direct correlation between CBF and CO. We hypothesized that measurement of CBF could be a good substitute for CO, even under more extreme hemodynamic conditions, for a wider range of critically ill patients. METHODS: Patients aged 65-80 years, undergoing elective cardiac surgery were included in this study. CBF in different cardiac cycles were measured by ultrasound: systolic carotid blood flow (SCF), diastolic carotid blood flow (DCF), and total (systolic and diastolic) carotid blood flow (TCF). CO simultaneously was measured by transesophageal echocardiography. RESULTS: For all patients, the correlation coefficients between SCF and CO, TCF and CO were 0.45 and 0.30, respectively, which were statistically significant, but not between DCF and CO. There was no significant correlation between either SCF, TCF or DCF and CO, when CO was <3.5 L/min. CONCLUSIONS: Systolic carotid blood flow may be used as a better index to replace CO. However, the method of direct measurement of CO is essential when the patient's heart function is poor.

Diagnostic value of ultrasound for predicting the occurrence of airway difficulty in patients undergoing anesthesia.

OBJECTIVE: To investigate the diagnostic value of ultrasound for predicting occurrence of airway difficulty in patients undergoing anesthesia. METHODS: A total of 273 patients airway difficultyundergoing general anesthesia admitted to the Department of Anesthesiology, Nanjing First Hospital, Affiliated to Nanjing Medical University from January 2017 to October 2021 were selected in this prospective study. Among them, 73 suffered airway difficulty and the airway difficultyremaining 200 did not. Factors relating to the occurrence of difficulty were observed, and the hyomental distance ratio [HMDR = hyomental distance at the extreme of head extension (HMDe)/hyomental distance in the neutral position (HMDn)] combined with the distance from skin to epiglottis midway (DSEM) were further studied for the prediction of airway difficulty occurrence. RESULTS: Multivariate regression analysis revealed that HMDe, HMDR, and DSEM were factors associated with the occurrence of difficulty (all P<0.05). The specificity and the sensitivity of HMDR in diagnosing airway difficulty were 0.715 and 0.918 respectively at a cutoff value of 1.245 mm. The specificity and sensitivity of DSEM in diagnosing airway difficulty were 0.959 and 0.767 respectively at a cutoff value of 22.952 nm. When HMDR was combined with DSEM, the specificity of the diagnosis of airway difficulty was 0.973, and the sensitivity was 0.904. CONCLUSION: HMDe, HMDR and DSEM can be used to predict occurrence of airway difficultyand HMDR combined with DSEM has value in the diagnosis.

The efficacy of ultrasonography on learning curve and inter-subject performance variability in radial artery cannulation in standardized training for residents: a randomized controlled trial.

AIM: To evaluate the effect of ultrasound (US) on learning curve and inter-subject performance variability of residents in radial artery cannulation. MATERIAL AND METHODS: Twenty non-anesthesiology residents who received standardized training in an anesthesiology department were selected and divided into two groups: anatomy group or US group. After training of relevant anatomy, US recognition and puncture skill, residents selected 10 patients either under US or anatomical localization performing radial artery catheterization. The number and time of successful cases of catheterization were recorded, success rate of first attempt and catheterization, as well as the total success rate of catheterization were calculated. The learning curve and inter-subject performance variability of residents were also calculated. Complications and the residents' satisfaction for teaching and self-confidence before puncture were also recorded. RESULTS: Compared to the anatomy group, total success rate and the success rate at first attempt were higher in US-guided group (88% vs. 57%, 94% vs. 81%). The average performance time in the US group was significantly less (2.9±0.8 min vs. 4.2±2.1 min) and the mean number of attempts was 1.6, while 2.6 for the anatomy group. With performing cases increasing, the average puncture time of residents in the US group decreased by 19s, while 14s in the anatomy group. More local hematoma occurred in the anatomy group. The satisfaction and confidence degree of residents were higher in US group ([98.5±6.5] vs [68.5±7.3], [90.2±8.6] vs [56.3±5.5]). CONCLUSION: US can significantly shorten the learning curve, reduce the inter-subject performance variability, improve the first attempt and total success rate of radial artery catheterization for non-anesthesiology residents.

Association Between Hypernatremia and Delirium After Cardiac Surgery: A Nested Case-Control Study.

Background: The association between hypernatremia and delirium after cardiac surgery has rarely been investigated. This study aimed to determine whether hypernatremia increases the risk of delirium after exposure. Materials and Methods: From April 2016 to June 2021, 7,831 consecutive patients receiving cardiac surgery were screened for potential enrollment. The primary outcome was postoperative delirium (POD). For the respective case of delirium, 10 controls were matched according to the index date within the nested case-control design. Hypernatremia exposure was defined as serum sodium > 145 mmol/L within 7 days before the index date. A generalized estimation equation was performed to assess excess risks for POD associated with hypernatremia, adjusted for demographics and clinical variables. Results: About 7,277 patients were included in the final analyses. About 669 (9.2%) patients with POD were assigned to the case group, and 6,690 controls were identified from the whole population. About 66.5% of the cases and 36.3% of the controls had hypernatremia exposure. After being adjusted to certain well-recognized confounding factors, hypernatremia showed a significant correlation with increased risk of delirium after cardiac surgery (adjusted OR, 1.73; 95% CI, 1.41~2.12). An e-value analysis suggested the robustness to unmeasured confounding. Conclusions: Hypernatremia was associated with an increased risk of delirium after cardiac surgery. This finding could have implications for risk stratification, early detection, and management of delirium in patients receiving cardiac surgery.

Intraoperative venous congestion rather than hypotension is associated with acute adverse kidney events after cardiac surgery: a retrospective cohort study.

BACKGROUND: The pathophysiological mechanisms by which venous congestion and hypotension lead to acute adverse kidney events after cardiac surgery with cardiopulmonary bypass have not been elucidated. We tested the hypothesis that intraoperative hypotension and venous congestion are associated with acute kidney injury and acute kidney disease. METHODS: Primary exposures were venous congestion and intraoperative hypotension defined by central venous pressure ≥12, 16, or 20 mm Hg or mean arterial pressure ≤55, 65, or 75 mm Hg. The primary outcomes were acute kidney injury and acute kidney disease. Multivariable logistic regression and Cox proportional hazard models were used, adjusted for relevant confounding factors and multiple comparisons. RESULTS: Of 5127 eligible subjects, 1070 (20.9%) and 327 (7.2%) developed acute kidney injury and acute kidney disease, respectively. The occurrence of acute kidney injury was statistically associated with both venous congestion and intraoperative hypotension. The cumulative incidence rate for new onset acute kidney disease was 1.34 (95% confidence interval [CI], 1.21-1.60) per 100 person-days. Acute kidney disease was significantly associated with each 10 min epoch of central venous pressure ≥12 mm Hg (hazard ratio [HR]=1.03; 99% CI, 1.01-1.06; P<0.001), ≥16 mm Hg (HR=1.04; 99% CI, 1.01-1.07; P<0.001), and ≥20 mm Hg (HR=1.07; 99% CI, 1.02-1.13; P<0.001). Venous congestion was associated with an 8-17% increased risk for de novo renal replacement therapy. In contrast, intraoperative hypotension was not associated with development of acute kidney disease. CONCLUSION: Although both venous congestion and intraoperative hypotension are associated with acute kidney injury, only venous congestion correlates with acute kidney disease among patients undergoing cardiac surgery requiring cardiopulmonary bypass. The reported associations are suggestive of a pathophysiological role of venous congestion in acute kidney disease.

Phosphatase and Tensin Homolog Deleted on Chromosome Ten Knockdown Attenuates Cognitive Deficits by Inhibiting Neuroinflammation in a Mouse Model of Perioperative Neurocognitive Disorder.

Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a crucial regulator of neuronal development, neuronal survival, axonal regeneration, and synaptic plasticity. In this study we examined the potential role of PTEN in cognitive function in a mouse model of perioperative neurocognitive disorder (PND). Adult male C57BL/6J mice received intracerebroventricular injections of small interfering RNA (siRNA) against PTEN or control siRNA 3 days prior to exploratory laparotomy (n = 8 per group). A group of healthy mice not undergoing surgery included as additional control. Barnes maze and fear conditioning tests were conducted 7 days after surgery. Mice were then sacrificed to examine the expression of PTEN, AMP-activated protein kinase (AMPK), ionized calcium binding adaptor molecule (Iba)-1, B-cell lymphoma (Bcl)-2, Bcl2-associated X protein (Bax), interleukin (IL)-1ß, and tumor necrosis factor (TNF)-α in the hippocampus. The microglial activation was examined by immunohistochemistry using Iba-1 as a microglia maker. Nissl and terminal transferase deoxyuridine triphosphate nick-end labeling (TUNEL) staining were used to measure cell death and apoptosis. In comparison to the healthy controls, surgically treated mice had longer latency to identify the target box in both training and testing sessions in the Barnes maze test and shorter freezing time in the fear conditioning test. Surgically treated mice had increased expression of PTEN, AMPK, Bax, IL-1ß, and TNF-α, as well as increasing number of activated microglia and apoptosis neurons in the hippocampus. PTEN knockdown significantly attenuated the behavioral deficits in Barnes maze and fear conditioning tests, as well as over-expression of PTEN, AMPK, Bax, IL-1ß, and TNF-α induced by surgery. PTEN knockdown could attenuate cognitive deficits induced by trauma, likely through inhibiting the activation of microglia.

Gastrodin alleviates perioperative neurocognitive dysfunction of aged mice by suppressing neuroinflammation.

Perioperative neurocognitive disorder (PND) is a common complication of elderly patients after surgery and lacks effective prevention and treatment measures. We investigated the effect and mechanism of gastrodin (GAS), a natural plant ingredient, on postoperative cognition induced by laparotomy in aged mice. Male aged (18 months) mice were subjected to laparotomy and orally treated with GAS (25, 50, and 100 mg/kg) 3 weeks before surgery and 1 week after surgery. In addition, some male aged (18 months) mice were subjected to viral vector or GSK-3ß expression virus injection followed by laparotomy with or without 100 mg/kg GAS treatment. GAS improved learning and memory in aged mice after surgery. Surgery increased the levels of pro-inflammatory factors (TNF-α, IL-1ß and IL-6) and decreased the level of an anti-inflammatory factor (IL-10) in the mouse hippocampus, and these changes were reversed by GAS treatment. GAS also suppressed the activation of microglia. GAS inhibited the phosphorylation of GSK-3ß and Tau. Furthermore, surgery induced more serious cognitive dysfunction, inflammatory factors, activation of microglia, and phosphorylation of GSK-3ß and Tau in GSK-3ß overexpressing aged mice. The improvement of learning and memory, the reduction of inflammation and microglia activation, and the suppression of GSK-3ß and Tau phosphorylation by GAS were prevented when GSK-3ß was overexpressed in aged mice subjected to surgery. Our finding suggested that GAS exerts neuroprotective effects in aged mice subjected to laparotomy by suppressing neuroinflammation and GSK-3ß and Tau phosphorylation. Thus, these findings suggest that GAS may be a promising agent for PND.

Chemokine CXCL13 acts via CXCR5-ERK signaling in hippocampus to induce perioperative neurocognitive disorders in surgically treated mice.

BACKGROUND: Perioperative neurocognitive disorders (PNDs) occur frequently after surgery and worsen patient outcome. How C-X-C motif chemokine (CXCL) 13 and its sole receptor CXCR5 contribute to PNDs remains poorly understood. METHODS: A PND model was created in adult male C57BL/6J and CXCR5-/- mice by exploratory laparotomy. Mice were pretreated via intracerebroventricular injection with recombinant CXCL13, short hairpin RNA against CXCL13 or a scrambled control RNA, or ERK inhibitor PD98059. Then surgery was performed to induce PNDs, and animals were assessed in the Barnes maze trial followed by a fear-conditioning test. Expression of CXCL13, CXCR5, and ERK in hippocampus was examined using Western blot, quantitative PCR, and immunohistochemistry. Levels of interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α) in hippocampus were assessed by Western blot. RESULTS: Surgery impaired learning and memory, and it increased expression of CXCL13 and CXCR5 in the hippocampus. CXCL13 knockdown partially reversed the effects of surgery on CXCR5 and cognitive dysfunction. CXCR5 knockout led to similar cognitive outcomes as CXCL13 knockdown, and it repressed surgery-induced activation of ERK and production of IL-1ß and TNF-α in hippocampus. Recombinant CXCL13 induced cognitive deficits and increased the expression of phospho-ERK as well as IL-1ß and TNF-α in hippocampus of wild-type mice, but not CXCR5-/- mice. PD98059 partially blocked CXCL13-induced cognitive dysfunction as well as production of IL-1ß and TNF-α. CONCLUSIONS: CXCL13-induced activation of CXCR5 may contribute to PNDs by triggering ERK-mediated production of pro-inflammatory cytokines in hippocampus.

Combination therapy with ropivacaine-loaded liposomes and nutrient deprivation for simultaneous cancer therapy and cancer pain relief.

Autophagy allows cancer cells to respond changes in nutrient status by degrading and recycling non-essential intracellular contents. Inhibition of autophagy combined with nutrient deprivation is an effective strategy to treat cancer. Pain is a primary determinant of poor quality of life in advanced cancer patients, but there is currently no satisfactory treatment. In addition, effective treatment of cancer does not efficiently relieve cancer pain, but may increase pain in many cases. Hence, few studies focus on simultaneous cancer therapy and pain relief, and made this situation even worse. Method: Ropivacaine was loaded into tumor-active targeted liposomes. The cytotoxicity of ropivacaine-based combination therapy in B16 and HeLa cells were tested. Moreover, a mice model of cancer pain which was induced by inoculation of melanoma near the sciatic nerve was constructed to assess the cancer suppression and pain relief effects of ropivacaine-based combination therapy. Results: Ropivacaine and ropivacaine-loaded liposomes (Rop-DPRL) were novelly found to damage autophagic degradation. Replicated administration of Rop-DPRL and calorie restriction (CR) could efficiently repress the development of tumor. In addition, administration of Rop-DPRL could relieve cancer pain with its own analgestic ability in a short duration, while repeated administration of Rop-DPRL and CR resulted in continuous alleviation of cancer pain through reduction of VEGF-A levels in advanced cancer mice. Further, dual inhibition of phosphorylation of STAT3 at Tyr705 and Ser727 by Rop-DPRL and CR contribute to the reduction of VEGF-A. Conclusion: Combination therapy with Rop-DPRL and nutrient deprivation simultaneously suppresses cancer growth and relieves cancer pain.

Overexpression of thymic stromal lymphopoietin is correlated with poor prognosis in epithelial ovarian carcinoma.

Thymic stromal lymphopoietin (TSLP) is an IL-7-like cytokine that has been reported to be associated with several malignant tumors. The present study aimed to evaluate its role in epithelial ovarian carcinoma (EOC). The mRNA levels of TSLP in human EOC samples and EOC cell lines were determined. Then, the expression of TSLP was examined in 144 clinical tissue microarray samples and correlated with clinicopathological factors. Finally, the correlation between TSLP overexpression and prognosis of EOC patients was analyzed. Our data show that mRNA levels of TSLP were significantly higher in EOC tissues and cell lines. Chi-square tests revealed that TSLP overexpression in EOC was significantly associated with age, histological type, Federation of Gynecology and Obstetrics (FIGO) stage, histological differentiation, pelvic involvement, and lymphatic metastasis. Kaplan-Meier survival analysis revealed that poor prognosis was significantly correlated with older age, advanced FIGO stage, poor histological differentiation, pelvic involvement, lymphatic involvement, or TSLP overexpression (P<0.05). Additionally, multivariate Cox regression analysis confirmed pelvic involvement and TSLP overexpression as independent prognostic factors for both overall and disease-free survival. Taken altogether, TSLP overexpression reflects a more malignant phenotype and TSLP may be a novel biomarker for EOC.

Dexmedetomidine Acts via the JAK2/STAT3 Pathway to Attenuate Isoflurane-Induced Neurocognitive Deficits in Senile Mice.

BACKGROUND: Previous studies showed that isoflurane-induced cognitive deficits could be alleviated by dexmedetomidine in young animal subjects. In the current study, we examine whether dexmedetomidine could also alleviate isoflurane-induced cognitive deficits in senile animals. METHODS: Senile male C57BL/6 mice (20 months) received dexmedetomidine (50 µg/kg, i.p.) or vehicle 30 minutes prior to isoflurane exposure (1.3% for 4 h). Cognitive function was assessed 19 days later using a 5-day testing regimen with Morris water maze. Some subjects also received pretreatment with α2 adrenoreceptor antagonist atipamezole (250 µg/kg, i.p.), JAK2 inhibitor AG490 (15 mg/kg i.p.) or STAT3 inhibitor WP1066 (40 mg/kg i.p.) 30 minutes prior to dexmedetomidine. RESULTS: Isoflurane exposure increased and reduced the time spent in the quadrant containing the target platform in training sessions. The number of crossings over the original target quadrant was also decreased. Dexmedotomidine attenuated such effects. Effects of dexmedotomidine were reduced by pretreatment with atipamezole, AG490 and WP1066. Increased phosphorylation of JAK2 and STAT3 in the hippocampus induced by isoflurane was augmented by dexmedetomidine. Effects of dexmedetomidine on JAK2/STAT3 phosphorylation were attenuated by atipamezole, AG490 and WP1066. Isoflurane promoted neuronal apoptosis and increased the expression of cleaved caspase-3 and BAD, and reduced Bcl-2 expression. Attenuation of such effects by dexmedotomidine was partially blocked by atipamezole, AG490 and WP1066. CONCLUSION: Dexmedetomidine could protect against isoflurane-induced spatial learning and memory impairment in senile mice by stimulating the JAK2/STAT3 signaling pathway. Such findings encourage the use of dexmedetomidine in geriatric patients receiving isoflurane anesthesia.

[Effects of dexmedetomidine on microcirculatory perfusion in rabbits with renal ischemia/reperfusion injury: quantitative evaluation with contrast-enhanced ultrasound].

OBJECTIVE: To investigate the effects of dexmedetomidine on renal microcirculatory perfusion in rabbits with renal ischemia/reperfusion (I/R) injury rabbit by quantitative analysis of contrast-enhanced ultrasound (CEUS). METHODS: Twenty- four New Zealand rabbits were randomly divided into 3 groups (8 in each), including a control group, renal I/R injury group and dexmedetomidine group. In the latter two groups, the right kidney of the rabbits was resected and I/R injury was induced in the left kidney. In dexmedetomidine group, the rabbits received an intraperitoneal dose of 10 µg/kg dexmedetomidine 30 min before renal ischemia, and 24 h after reperfusion, the renal size and renal artery resistance (RI) were measured, and renal cortex perfusion was observed by CEUS. The time-to-peak intensity (TTP), peak signal intensity (PSI), gradient between start frame to peak frame (Grad) and area under the curve (AUC) were quantitatively analyzed using the time-intensity curves. Pathological changes of the kidney were also observed. RESULTS: Compared with the control group, the rabbits in I/R and dexmedetomidine groups showed distinct changes of the renal size with obvious renal pathologies. RI, PPT and AUC all increased, and PSI and Grad decreased significantly in I/R and dexmedetomidine groups (P<0.05). Compared with I/R group, obvious improvement of the renal size and renal pathologies were observed in dexmedetomidine group, which showed significantly decreased RI, PPT and AUC and increased PSI and Grad (P<0.05). CONCLUSION: CEUS combined with the time-intensity curve parameters allows quantitative and dynamic analysis of the protective effects of dexmedetomidine on microcirculatory perfusion in rabbits with renal I/R injury.

A neurosteroid analogue photolabeling reagent labels the colchicine-binding site on tubulin: a mass spectrometric analysis.

Previous studies have shown that the neurosteroid analogue, 6-Azi-pregnanolone (6-AziP), photolabels voltage-dependent anion channels and proteins of approximately 55 kDa in rat brain membranes. The present study used two-dimensional electrophoresis and nanoelectrospray ionization ion-trap mass spectrometry (nano-ESI-MS) to identify the 55 kDa proteins (isoelectric point 4.8) as isoforms of ß-tubulin. This identification was confirmed by immunoblot and immunoprecipitation of photolabeled protein with anti-ß-tubulin antibody and by the demonstration that 6-AziP photolabels purified bovine brain tubulin in a concentration-dependent pattern. To identify the photolabeling sites, purified bovine brain tubulin was photolabeled with 6-AziP, digested with trypsin, and analyzed by matrix-assisted laser desorption/ionization MS (MALDI). A 6-AziP adduct of TAVCDIPPR(m/z = 1287.77), a ß-tubulin specific peptide, was detected by MALDI. High-resolution liquid chromatography-MS/MS analysis identified that 6-AziP was covalently bound to cysteine 354 (Cys-354), previously identified as a colchicine-binding site. 6-AziP photolabeling was inhibited by 2-methoxyestradiol, an endogenous derivative of estradiol thought to bind to the colchicine site. Structural modeling predicted that neurosteroids could dock in this colchicine site at the interface between α- and ß-tubulin with the photolabeling group of 6-AziP positioned proximate to Cys-354.