Tanshinone IIA Regulates Synaptic Plasticity in Mg2+-Free-Induced Epileptic Hippocampal Neurons via the PI3K/Akt Signaling Pathway.

BACKGROUND: Tanshinone IIA (TSIIA) is an element of the effective ingredients of Salvia miltiorrhiza Bunge (Labiatae), exhibits a significant therapeutic effect in brain neuroprotection. The focus of this study was the examination of synaptic plasticity of in Mg2+-free-induced epileptic hippocampus neurons and how TSIIA protects against it. METHODS: The purity of the primary hippocampal neurons extracted from Sprague Dawley rats was assessed within 24 hours by microtubule-associated protein (MAP2) immunofluorescence staining. A hippocampal neuron model for Mg2+-free-induced spontaneous recurrent epileptiform discharge was developed, five experimental groups were then randomized: blank (Blank), model (Model), TSIIA (TSIIA, 20 µM), LY294002 (LY294002, 25 µM), and TSIIA+LY294002 (TSIIA+LY294002, 20 µM+25 µM). FIJI software was used to examine variations of neurite complexity, total length of hippocampal neurons, number of primary dendrites and density of dendritic spines. Developmental regulation brain protein (Drebrin) and brain-derived neurotrophic factor (BDNF) expression was evaluated using immunofluorescence staining and the relative expression of phospho-protein kinase B (p-Akt)/Akt, BDNF, synaptophysin (SYN) and postsynaptic density 95 (PSD-95) determined by Western blot. RESULTS: In contrast to the model group, TSIIA drastically reduced damage to synaptic plasticity of hippocampal neurons caused by epilepsy (p < 0.05). The TSIIA group showed a significant increase in the relative expression of PSD-95, SYN, BDNF, and p-Akt/Akt (p < 0.01). CONCLUSIONS: TSIIA was effective in reducing harm to the synaptic plasticity of hippocampal neurons induced by persistent status epilepticus, with the possible mechanism being regulation of the phosphatidylinositol 3-kinase 56 (PI3K)/Akt signaling pathway.

Clot-based radiomics model for cardioembolic stroke prediction with CT imaging before recanalization: a multicenter study.

OBJECTIVES: To develop a clot-based radiomics model using CT imaging radiomic features and machine learning to identify cardioembolic (CE) stroke before mechanical thrombectomy (MTB) in patients with acute ischemic stroke (AIS). MATERIALS AND METHODS: This retrospective four-center study consecutively included 403 patients with AIS who sequentially underwent CT and MTB between April 2016 and July 2021. These were grouped into training, testing, and external validation cohorts. Thrombus-extracted radiomic features and basic information were gathered to construct a machine learning model to predict CE stroke. The radiological characteristics and basic information were used to build a routine radiological model. A combined radiomics and radiological features model was also developed. The performances of all models were evaluated and compared in the validation cohort. A histological analysis helped further assess the proposed model in all patients. RESULTS: The radiomics model yielded an area under the curve (AUC) of 0.838 (95% confidence interval [CI], 0.771-0.891) for predicting CE stroke in the validation cohort, significantly higher than the radiological model (AUC, 0.713; 95% CI, 0.636-0.781; p = 0.007) but similar to the combined model (AUC, 0.855; 95% CI, 0.791-0.906; p = 0.14). The thrombus radiomic features achieved stronger correlations with red blood cells (|rmax|, 0.74 vs. 0.32) and fibrin and platelet (|rmax|, 0.68 vs. 0.18) than radiological characteristics. CONCLUSION: The proposed CT-based radiomics model could reliably predict CE stroke in AIS, performing better than the routine radiological method. KEY POINTS: • Admission CT imaging could offer valuable information to identify the acute ischemic stroke source by radiomics analysis. • The proposed CT imaging-based radiomics model yielded a higher area under the curve (0.838) than the routine radiological method (0.713; p = 0.007). • Several radiomic features showed significantly stronger correlations with two main thrombus constituents (red blood cells, |rmax|, 0.74; fibrin and platelet, |rmax|, 0.68) than routine radiological characteristics.

Elucidation of N-/O-glycosylation and site-specific mapping of sialic acid linkage isomers of SARS-CoV-2 human receptor angiotensin-converting enzyme 2.

Human angiotensin-converting enzyme 2 (hACE2) is the primary receptor for cellular entry of SARS-CoV-2 into human host cells. hACE2 is heavily glycosylated and glycans on the receptor may play a role in viral binding. Thus, comprehensive characterization of hACE2 glycosylation could aid our understanding of interactions between the receptor and SARS-CoV-2 spike (S) protein, as well as provide a basis for the development of therapeutic drugs targeting this crucial interaction. Herein, 138 N-glycan compositions were identified, most of which are complex-type N-glycans, from seven N-glycosites of hACE2. Among them, 67% contain at least one sialic acid residue. At the level of glycopeptides, the overall quantification of sialylated glycan isomers observed on the sites N322 and N546 have a higher degree of NeuAc (α2-3)Gal (over 80.3%) than that of other N-glycosites (35.6-71.0%). In terms of O-glycans, 69 glycan compositions from 12 O-glycosites were identified, and especially, the C-terminus of hACE2 is heavily O-glycosylated. The terminal sialic acid linkage type of H1N1S1 and H1N1S2 are covered highly with α2,3-sialic acid. These findings could aid the investigation of the interaction between SARS-CoV-2 and human host cells.

Investigation of the mechanism of tanshinone IIA to improve cognitive function via synaptic plasticity in epileptic rats.

CONTEXT: Tanshinone IIA is an extract of Salvia miltiorrhiza Bunge (Labiatae) used to treat cardiovascular disorders. It shows potential anticonvulsant and cognition-protective properties. OBJECTIVE: We investigated the mechanism of tanshinone IIA on antiepileptic and cognition-protective effects in the model of epileptic rats. MATERIALS AND METHODS: Lithium chloride (LiCl)-pilocarpine-induced epileptic Wistar rats were randomly assigned to the following groups (n = 12): control (blank), model, sodium valproate (VPA, 189 mg/kg/d, positive control), tanshinone IIA low dose (TS IIA-L, 10 mg/kg/d), medium dose (TS IIA-M, 20 mg/kg/d) and high dose (TS IIA-H, 30 mg/kg/d). Then, epileptic behavioural observations, Morris water maze test, Timm staining, transmission electron microscopy, immunofluorescence staining, western blotting and RT-qPCR were measured. RESULTS: Compared with the model group, tanshinone IIA reduced the frequency and severity of seizures, improved cognitive impairment, and inhibited hippocampal mossy fibre sprouting score (TS IIA-M 1.50 ± 0.22, TS IIA-H 1.17 ± 0.31 vs. model 2.83 ± 0.31), as well as improved the ultrastructural disorder. Tanshinone IIA increased levels of synapse-associated proteins synaptophysin (SYN) and postsynaptic dense substance 95 (PSD-95) (SYN: TS IIA 28.82 ± 2.51, 33.18 ± 2.89, 37.29 ± 1.69 vs. model 20.23 ± 3.96; PSD-95: TS IIA 23.10 ± 0.91, 26.82 ± 1.41, 27.00 ± 0.80 vs. model 18.28 ± 1.01). DISCUSSION AND CONCLUSIONS: Tanshinone IIA shows antiepileptic and cognitive function-improving effects, primarily via regulating synaptic plasticity. This research generates a theoretical foundation for future research on potential clinical applications for tanshinone IIA.

Overexpression of GhABF3 increases cotton(Gossypium hirsutum L.) tolerance to salt and drought.

BACKGROUND: Plants suffer from various abiotic stresses during their lifetime, of which drought and salt stresses are two main factors limiting crop yield and quality. Previous studies have shown that abscisic acid (ABA) responsive element binding protein (AREB)/ ABRE binding factors (ABFs) in bZIP transcription factors are involved in plant stress response in an ABA-dependent manner. However, little is known about the properties and functions of AREB/ABFs, especially ABF3, in cotton. RESULTS: Here, we reported the cloning and characterization of GhABF3. Expression of GhABF3 was induced by drought,salt and ABA treatments. Silencing of GhABF3 sensitized cotton to drought and salt stress, which was manifested in decreased cellular antioxidant capacity and chlorophyll content. Overexpression of GhABF3 significantly improved the drought and salinity tolerance of Arabidopsis and cotton. Exogenous expression of GhABF3 resulted in longer root length and less leaf wilting under stress conditions in Arabidopsis thaliana. Overexpressing GhABF3 significantly improved salt tolerance of upland cotton by reducing the degree of cellular oxidation, and enhanced drought tolerance by decreasing leaf water loss rate. The increased expression of GhABF3 up-regulated the transcriptional abundance of downstream ABA-inducible genes under salt stress in Arabidopsis. CONCLUSION: In conclusion, our results demonstrated that GhABF3 plays an important role in plant drought and salt tolerance. Manipulation of GhABF3 by biotechnology might be an important strategy to alter the stress resistance of cotton.

Relative Quantification of N-Glycopeptide Sialic Acid Linkage Isomers by Ion Mobility Mass Spectrometry.

Sialic acids decorate the surface of glycoproteins and play important roles in a variety of pathological processes. Although the mass spectrometry (MS) based linkage-specific analysis of sialylated N-glycopeptide is developing rapidly, quantitative analysis of these isomers still remains a challenge. Herein, we reported a novel quantitative strategy that can unambiguously identify and relatively quantify linkage-specific N-glycopeptides using ion mobility mass spectrometry (IM-MS). Without the assistance of derivatization, this method can relatively quantify sialic acid isomers of intact glycopeptides by using their characteristic fragment ions in IM-MS. Moreover, good linearity (R2 > 0.99) of relative quantification within a dynamic range of 2 orders of magnitude and high reproducibility (coefficient of variation (CV) < 10%, n = 3) were demonstrated. Finally, our results illustrated the aberrant sialylation of haptoglobin (Hp) in hepatocellular carcinoma (HCC), where the ratios of α2,3 to α2,6 sialylation of seven N-glycopeptides were found to be significantly altered (p < 0.01) in HCC individuals (n = 27) compared with healthy controls (n = 27).

Visualization of Thrombus Enhancement on Thin-Slab Maximum Intensity Projection of CT Angiography: An Imaging Sign for Predicting Stroke Source and Thrombus Compositions.

Background Thrombus enhancement (TE) in large vessel occlusion in patients with acute ischemic stroke can be visualized with thin-slab maximum intensity projection (TS-MIP) image reconstruction of CT angiograms. Purpose To evaluate whether TE on TS-MIP reconstructed CT angiograms can be used to predict thrombus composition and stroke source. Materials and Methods This retrospective study included consecutive patients with acute ischemic stroke who underwent thrombectomy in the anterior circulation between August 2016 and July 2019. Stroke types were classified according to the Trial of ORG 10172 in Acute Stroke Treatment. TE on TS-MIP reconstructed CT angiograms was evaluated by two readers. Various clinical and interventional parameters and histopathologic thrombi examination results were compared between the TE-positive and TE-negative groups. The associations between TE and thrombus compositions and stroke sources were analyzed by using multivariable linear and logistic regression models. Results A total of 148 patients (mean age, 71 years ± 11 [standard deviation]; 94 men) were included. TE was confirmed in 80% (119 of 148) of the patients. TE-positive thrombi contained a higher fibrin and platelet proportion (mean, 46% ± 16 vs 34% ± 13; P = .02) and fewer erythrocytes (mean, 33% ± 14 vs 48% ± 20, P = .002) than the TE-negative thrombi. The proportion of cardioembolic and cryptogenic strokes in the TE-positive and TE-negative groups was 92% (110 of 119) and 24% (seven of 29), respectively (P < .001). In adjusted analysis, the presence of TE (odds ratio, 155; 95% CI: 17, 1438; P < .001) was associated with a combination of cardioembolic and cryptogenic strokes. A multiple logistic regression model showed that TE (odds ratio, 23; 95% CI: 1.8, 288; P = .02) was significantly associated with cardioembolic stroke. Conclusion Thrombus enhancement on thin-slab maximum intensity projection of CT angiography can be used to predict cardioembolic and cryptogenic strokes and identify thrombi with a higher fibrin-to-platelet fraction and a lower erythrocyte proportion. © RSNA, 2020 Online supplemental material is available for this article. See also the editorial by Kansagra and Goyal in this issue.

Simultaneous Quantification of Protein Expression and Modifications by Top-down Targeted Proteomics: A Case of the Sarcomeric Subproteome.

Determining changes in protein expression and post-translational modifications (PTMs) is crucial for elucidating cellular signal transduction and disease mechanisms. Conventional antibody-based approaches have inherent problems such as the limited availability of high-quality antibodies and batch-to-batch variation. Top-down mass spectrometry (MS)-based proteomics has emerged as the most powerful method for characterization and quantification of protein modifications. Nevertheless, robust methods to simultaneously determine changes in protein expression and PTMs remain lacking. Herein, we have developed a straightforward and robust top-down liquid chromatography (LC)/MS-based targeted proteomics platform for simultaneous quantification of protein expression and PTMs with high throughput and high reproducibility. We employed this method to analyze the sarcomeric subproteome from various muscle types of different species, which successfully revealed skeletal muscle heterogeneity and cardiac developmental changes in sarcomeric protein isoform expression and PTMs. As demonstrated, this targeted top-down proteomics platform offers an excellent 'antibody-independent' alternative for the accurate quantification of sarcomeric protein expression and PTMs concurrently in complex mixtures, which is generally applicable to different species and various tissue types.

Novel Sarcopenia-related Alterations in Sarcomeric Protein Post-translational Modifications (PTMs) in Skeletal Muscles Identified by Top-down Proteomics.

Sarcopenia, the age-related loss of skeletal muscle mass and strength, is a significant cause of morbidity in the elderly and is a major burden on health care systems. Unfortunately, the underlying molecular mechanisms in sarcopenia remain poorly understood. Herein, we utilized top-down proteomics to elucidate sarcopenia-related changes in the fast- and slow-twitch skeletal muscles of aging rats with a focus on the sarcomeric proteome, which includes both myofilament and Z-disc proteins-the proteins that constitute the contractile apparatuses. Top-down quantitative proteomics identified significant changes in the post-translational modifications (PTMs) of critical myofilament proteins in the fast-twitch skeletal muscles of aging rats, in accordance with the vulnerability of fast-twitch muscles to sarcopenia. Surprisingly, age-related alterations in the phosphorylation of Cypher isoforms, proteins that localize to the Z-discs in striated muscles, were also noted in the fast-twitch skeletal muscle of aging rats. This represents the first report of changes in the phosphorylation of Z-disc proteins in skeletal muscle during aging. In addition, increased glutathionylation of slow skeletal troponin I, a novel modification that may help protect against oxidative damage, was observed in slow-twitch skeletal muscles. Furthermore, we have identified and characterized novel muscle type-specific proteoforms of myofilament proteins and Z-disc proteins, including a novel isoform of the Z-disc protein Enigma. The finding that the phosphorylation of Z-disc proteins is altered in response to aging in the fast-twitch skeletal muscles of aging rats opens new avenues for the investigation of the role of Z-discs in age-related muscle dysfunction.

Electrophilic probes for deciphering substrate recognition by O-GlcNAc transferase.

O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential human glycosyltransferase that adds O-GlcNAc modifications to numerous proteins. However, little is known about the mechanism with which OGT recognizes various protein substrates. Here we report on GlcNAc electrophilic probes (GEPs) to expedite the characterization of OGT-substrate recognition. Data from mass spectrometry, X-ray crystallization, and biochemical and radiolabeled kinetic assays support the application of GEPs to rapidly report the impacts of OGT mutations on protein substrate or sugar binding and to discover OGT residues crucial for protein recognition. Interestingly, we found that the same residues on the inner surface of the N-terminal domain contribute to OGT interactions with different protein substrates. By tuning reaction conditions, a GEP enables crosslinking of OGT with acceptor substrates in situ, affording a unique method to discover genuine substrates that weakly or transiently interact with OGT. Hence, GEPs provide new strategies to dissect OGT-substrate binding and recognition.

Evaluation of Quiescent-Interval Single-Shot Magnetic Resonance Angiography in Diabetic Patients With Critical Limb Ischemia Undergoing Digital Subtraction Angiography: Comparison With Contrast-Enhanced Magnetic Resonance Angiography With Calf Compression at 3.0 Tesla.

PURPOSE: To assess the diagnostic performance of quiescent-interval single-shot magnetic resonance angiography (QISS-MRA) at 3 tesla in diabetic patients with critical limb ischemia (CLI) vs contrast-enhanced MR angiography (CE-MRA) using digital subtraction angiography (DSA) as the standard of reference. METHOD: Thirty-seven consecutive diabetic patients (mean age 71.8±7.2 years; 30 men) with CLI (Fontaine stage III-IV) underwent QISS-MRA and CE-MRA with calf compression; DSA was the standard. Image quality (5-point Likert-type scale) and stenosis severity (5-point grading) for QISS-MRA and CE-MRA were evaluated by 2 blinded readers in 1147 and 654 vessel segments, respectively. Per-segment and per-region (pelvis, thigh, calf) sensitivity, specificity, positive predictive value, and negative predictive value were calculated. RESULTS: Image quality of QISS-MRA was lower compared with CE-MRA in the pelvic region (p<0.001 in both readers) and thigh region (p=0.033 in reader 1 and p=0.018 in reader 2), whereas in the calf region, the image quality of QISS-MRA was better than CE-MRA (p=0.009 in reader 1 and p=0.001 in reader 2). In segment-based analyses, there was no difference between QISS-MRA and CE-MRA in sensitivity [89.5% vs 90.3% in reader 1 (p=0.774) and 87.6% vs 90.6% in reader 2 (p=0.266)] or specificity [94.2% vs 92.9% in reader 1 (p=0.513) and 92.9% vs 92.9% in reader 2 (p>0.999)]. In region-based analyses, QISS-MRA and CE-MRA yielded similar sensitivity and specificity in all areas but the pelvic region for reader 2 (specificity 95.5% vs 84.8%, p=0.041). CONCLUSION: QISS-MRA performed very well in diabetic patients with CLI and was a good alternative for patients with contraindications to CE-MRA.

Purification, Characterization and Degradation Performance of a Novel Dextranase from Penicillium cyclopium CICC-4022.

A novel dextranase was purified from Penicillium cyclopium CICC-4022 by ammonium sulfate fractional precipitation and gel filtration chromatography. The effects of temperature, pH and some metal ions and chemicals on dextranase activity were investigated. Subsequently, the dextranase was used to produce dextran with specific molecular mass. Weight-average molecular mass (Mw) and the ratio of weight-average molecular mass/number-average molecular mass, or polydispersity index (Mw/Mn), of dextran were measured by multiple-angle laser light scattering (MALS) combined with gel permeation chromatography (GPC). The dextranase was purified to 16.09-fold concentration; the recovery rate was 29.17%; and the specific activity reached 350.29 U/mg. Mw of the dextranase was 66 kDa, which is similar to dextranase obtained from other Penicillium species reported previously. The highest activity was observed at 55 °C and a pH of 5.0. This dextranase was identified as an endodextranase, which specifically degraded the α-1,6 glucosidic bonds of dextran. According to metal ion dependency tests, Li⁺, Na⁺ and Fe2+ were observed to effectively improve the enzymatic activity. In particular, Li⁺ could improve the activity to 116.28%. Furthermore, the dextranase was efficient at degrading dextran and the degradation rate can be well controlled by the dextranase activity, substrate concentration and reaction time. Thus, our results demonstrate the high potential of this dextranase from Penicillium cyclopium CICC-4022 as an efficient enzyme to produce specific clinical dextrans.

Neuronal protective effect of Songling Xuemaikang capsules alone and in combination with carbamazepine on epilepsy in kainic acid-kindled rats.

CONTEXT: Epilepsy is a common life-threatening neurological disorder that is often drug-resistant and associated with cognitive impairment. The traditional Chinese patent medicine Songling Xuemaikang capsules (SXC) is clinically used for epilepsy therapy and alleviation of cognitive impairment. OBJECTIVE: This study investigates the neuronal protective effect of SXC combined with carbamazepine (CBZ) on epilepsy and cognitive impairment in kainic acid-kindled SD rats. MATERIALS AND METHODS: Kainic acid-kindled rats were established by injection of 0.45 µg kainic acid and randomly divided into 5 groups (n = 14): saline (sham-operated), control, CBZ, SXC and CBZ + SXC combined group. Rats in the treatment groups received CBZ (50 mg/kg/d), SXC (600 mg/kg/d) or combined CBZ (50 mg/kg/d) + SXC (600 mg/kg/d) via intragastric injection for 60 days. Epileptic behaviours, cognitive impairment, neuronal apoptosis and expression of p-Akt, Akt and caspase-9 were measured, and the alleviation of cognitive damage and neuronal apoptosis was analyzed. RESULTS: The combined administration of SXC and CBZ significantly decreased the frequency of seizures (1.2 ± 0.3) and the number of episodes (1.3 ± 0.5) above stage III (p < 0.05). Neuronal apoptosis was improved (p < 0.01), and cognitive damage was ameliorated (p < 0.05).The level of p-Akt was enhanced (p < 0.01) whereas the expression of caspase-9 was evidently inhibited (p < 0.01) in the combined group. CONCLUSIONS: The present findings confirm that the combined use of SXC with CBZ can effectively control epileptic seizures, alleviate damage to hippocampal neurons and protect against cognitive impairment. The mechanism of action might be related to the upregulation of p-Akt and inhibition of caspase-9 expression.

Morphological characteristics of chronic total occlusion: predictors of different strategies for long-segment femoral arterial occlusions.

OBJECTIVES: To investigate morphological characteristics used to predict recanalisation strategies in long-segment (>10 cm) femoral chronic total occlusion (LSF-CTO) angioplasty. METHODS: We retrospectively evaluated a range of morphological CTA and DSA features in patients who underwent recanalisation of LSF-CTO. The stage of CTO was classified into early (3-12 months) and late (>12 months) according to estimated duration. Characteristics including stump morphology, lesion length and calcification, proximal side branches, collaterals circulation, runoff vessels and concomitant arterial occlusion were used as predictors, and multivariate logistic regression analysis was performed to identify variables associated with late-stage CTO and retrograde technique. RESULTS: A total of 119 patients with 137 CTOs in 137 limbs were enrolled. Overall, successful recanalisation was achieved in 122 CTOs (89.1%). Flush occlusion [odds ratio (OR) 2.958; 95% confidence interval (CI) 1.172-7.465; p = 0.022], large collateral (OR 2.778; 95% CI 1.201-6.427; p = 0.017) and TransAtlantic Inter-Society Consensus II class D (TASC D) lesion (OR 1.743; 95% CI 1.019-2.981; p = 0.042) were predictors for late-stage CTO. Flush occlusion (OR 75.278; 95% CI 10.664-531.384; p < 0.001) and large collateral (OR 23.213; 95% CI 3.236-166.523; p = 0.002) were associated with high likelihood for retrograde approach. CONCLUSIONS: Flush occlusion and large collateral were associated with a CTO at late-stage which may require retrograde recanalisation. KEY POINTS: • CTO morphological characteristics help estimate lesion duration and optimise recanalisation strategies. • Flush occlusion and large collateral is associated with late-stage CTO and retrograde recanalisation. • Application of anterograde and retrograde recanalisation for long-segment femoral CTO is effective.

Correction to: Morphological characteristics of chronic total occlusion: predictors of different strategies for long-segment femoral arterial occlusions.

The original version of this article unfortunately contained mistakes. The legends to Figs. 2-4 were incorrectly interchanged. The correct versions are given below. The original article has been corrected.

Protective effect of compound Danshen (Salvia miltiorrhiza) dripping pills alone and in combination with carbamazepine on kainic acid-induced temporal lobe epilepsy and cognitive impairment in rats.

CONTEXT: Temporal lobe epilepsy (TLE) is resistant to antiepileptic drugs (AEDs) and is associated with cognitive impairment. The modern Chinese medicine, compound Danshen dripping pills (CDDP), is clinically effective in treating epilepsy and improving cognitive impairment. OBJECTIVE: This study evaluated the protective effects of CDDP alone and in combination with carbamazepine (CBZ) on kainic acid-induced TLE and cognitive impairment in rats. MATERIALS AND METHODS: Sprague-Dawley rats were randomly divided into five groups: control (sham operated), model, CDDP, CBZ and combined. A TLE model was then created via bilateral intrahippocampal injection of 0.35 µg kainic acid (KA). Rats received CDDP (85 mg/kg), CBZ (100 mg/kg) or combined (85 mg/kg CDDP +100 mg/kg CBZ) via intragastric administration for 90 d, respectively. Seizure intensity, apoptosis and glial cell line-derived neurotrophic factor (GDNF) were measured. Furthermore, the improvement in cognitive impairment and hippocampal neuronal damage was evaluated. RESULTS: CDDP combined with CBZ significantly decreased seizure severity and frequency (p < 0.05) and ameliorated cognitive impairment (p < 0.05). The model group showed a significant reduction of neurons and Bcl-2/Bax expression in the hippocampus CA3 area (p < 0.01), the combined groups significantly reversed these change (p < 0.01). GDNF expression in the combined groups showed a clear increase over the model group (p < 0.05). CONCLUSION: These findings support the use of CDDP as an adjuvant drug for the treatment of TLE and cognitive deficit. Its mechanism might be related to an anti-apoptosis effect and up-regulation of GDNF.

Complete Characterization of Cardiac Myosin Heavy Chain (223 kDa) Enabled by Size-Exclusion Chromatography and Middle-Down Mass Spectrometry.

Myosin heavy chain (MHC), the major component of the myosin motor molecule, plays an essential role in force production during muscle contraction. However, a comprehensive analysis of MHC proteoforms arising from sequence variations and post-translational modifications (PTMs) remains challenging due to the difficulties in purifying MHC (∼223 kDa) and achieving complete sequence coverage. Herein, we have established a strategy to effectively purify and comprehensively characterize MHC from heart tissue by combining size-exclusion chromatography (SEC) and middle-down mass spectrometry (MS). First, we have developed a MS-compatible SEC method for purifying MHC from heart tissue with high efficiency. Next, we have optimized the Glu-C, Asp-N, and trypsin limited digestion conditions for middle-down MS. Subsequently, we have applied this strategy with optimized conditions to comprehensively characterize human MHC and identified ß-MHC as the predominant isoform in human left ventricular tissue. Full sequence coverage based on highly accurate mass measurements has been achieved using middle-down MS combining 1 Glu-C, 1 Asp-N, and 1 trypsin digestion. Three different PTMs: acetylation, methylation, and trimethylation were identified in human ß-MHC and the corresponding sites were localized to the N-terminal Gly, Lys34, and Lys129, respectively, by electron capture dissociation (ECD). Taken together, we have demonstrated this strategy is highly efficient for purification and characterization of MHC, which can be further applied to studies of the role of MHC proteoforms in muscle-related diseases. We also envision that this integrated SEC/middle-down MS strategy can be extended for the characterization of other large proteins over 200 kDa.

Technical feasibility and tissue reaction after silicone-covered biodegradable magnesium stent insertion in the oesophagus: a primary study in vitro and in vivo.

OBJECTIVES: Determine the feasibility of and tissue response to biodegradable magnesium-silicone stent insertion into the oesophagus of rabbits. METHODS: Mechanical compression-recovery and degradation behaviours of the stents were investigated in vitro. Thirty rabbits were randomly divided into a magnesium-silicone stent group (n = 15) that received stent insertion into the lower 1/3 of the oesophagus under fluoroscopic guidance and a control group (n = 15). Oesophagography was performed at 1, 2 and 4 weeks. Five rabbits in each group were euthanized at each time point for histological examination. RESULTS: Magnesium-silicone stents showed good flexibility and elasticity, and degraded more slowly than bare stents at pH 4.0 and 7.4. All stent insertions were well tolerated. The oesophageal diameters at 1, 2 and 4 weeks were 9.7 ± 0.7, 9.6 ± 0.8 and 9.6 ± 0.5 mm, respectively (vs. 9.2 ± 0.8 mm before intervention; P > 0.05). Stent migration occurred in six rabbits (one at 1 week, one at 2 and four at 4). Microscopy demonstrated dilation of the oesophageal wall within 1 week of insertion. Oesophageal injury and collagen deposition following stent insertion were similar to control (P > 0.05). CONCLUSIONS: Oesophageal magnesium-silicone stent insertion was feasible and provided reliable support for 2 weeks without causing oesophageal injury or collagen deposition. KEY POINTS: • Mg stent provided apparently adequate radial force and silicone membrane reduced magnesium biodegradation • Stent insertion provided good support for at least 2 weeks before biodegradation • Stenting effectively resulted in oesophageal wall remodelling, without demonstrable injury.

String-like lumen in below-the-knee chronic total occlusions on contrast-enhanced magnetic resonance angiography predicts intraluminal recanalization and better blood flow restoration.

OBJECTIVES: To determine whether string-like lumina (SLs) on contrast-enhanced magnetic resonance angiography (CE-MRA) predict better outcomes in diabetic patients with below-the-knee (BTK) chronic total occlusions (CTOs). METHODS: This study involved 317 long-segment (>5 cm) BTK CTOs of 245 patients that were examined using CE-MRA and treated using endovascular angioplasty. An SL with a CTO was slowly filled with blood on conventional CE-MRA. Univariate and multivariate analyses were performed to identify predictors of procedural success, recanalisation method and immediate blood flow restoration. The target-lesion patency and limb-salvage rates were assessed. RESULTS: SL-positive CTOs (n = 60) achieved a higher technique success rate, preferred intraluminal angioplasty and better blood flow restoration than SL-negative CTOs (n = 257, P < 0.05). Multivariate analyses revealed that lesion length was the independent predictor of procedural success (P = 0.028). SL was a predictor of intraluminal angioplasty (P < 0.001) and good blood-flow restoration (P = 0.004). Kaplan-Meier analyses at 12 months revealed a higher target lesion patency rate (P = 0.04) and limb-salvage rate (P = 0.35) in SL-positive CTOs. CONCLUSIONS: In patients with BTK CTOs, SL predicted intraluminal angioplasty and good blood-flow restoration for BTK CTOs. KEY POINTS: • Intraluminal recanalisation was more frequently used for BTK-CTOs with SLs than without • CTO length was the only independent predictor of successful CTO recanalisation • SL was the only predictor of intraluminal angioplasty for BTK-CTOs • SL and CTO length were predictors of good blood-flow restoration after recanalisation • Restenosis-free and limb-salvage rates were better for SL-positive CTOs than SL-negative CTOs.

Top-Down Targeted Proteomics Reveals Decrease in Myosin Regulatory Light-Chain Phosphorylation That Contributes to Sarcopenic Muscle Dysfunction.

Sarcopenia, the loss of skeletal muscle mass and function with advancing age, is a significant cause of disability and loss of independence in the elderly and thus, represents a formidable challenge for the aging population. Nevertheless, the molecular mechanism(s) underlying sarcopenia-associated muscle dysfunction remain poorly understood. In this study, we employed an integrated approach combining top-down targeted proteomics with mechanical measurements to dissect the molecular mechanism(s) in age-related muscle dysfunction. Top-down targeted proteomic analysis uncovered a progressive age-related decline in the phosphorylation of myosin regulatory light chain (RLC), a critical protein involved in the modulation of muscle contractility, in the skeletal muscle of aging rats. Top-down tandem mass spectrometry analysis identified a previously unreported bis-phosphorylated proteoform of fast skeletal RLC and localized the sites of decreasing phosphorylation to Ser14/15. Of these sites, Ser14 phosphorylation represents a previously unidentified site of phosphorylation in RLC from fast-twitch skeletal muscle. Subsequent mechanical analysis of single fast-twitch fibers isolated from the muscles of rats of different ages revealed that the observed decline in RLC phosphorylation can account for age-related decreases in the contractile properties of sarcopenic fast-twitch muscles. These results strongly support a role for decreasing RLC phosphorylation in sarcopenia-associated muscle dysfunction and suggest that therapeutic modulation of RLC phosphorylation may represent a new avenue for the treatment of sarcopenia.