Rasal1 regulates calcium dependent neuronal maturation by modifying microtubule dynamics.

BACKGROUND: Rasal1 is a Ras GTPase-activating protein which contains C2 domains necessary for dynamic membrane association following intracellular calcium elevation. Membrane-bound Rasal1 inactivates Ras signaling through its RasGAP activity, and through such mechanisms has been implicated in regulating various cellular functions in the context of tumors. Although highly expressed in the brain, the contribution of Rasal1 to neuronal development and function has yet to be explored. RESULTS: We examined the contributions of Rasal1 to neuronal development in primary culture of hippocampal neurons through modulation of Rasal1 expression using molecular tools. Fixed and live cell imaging demonstrate diffuse expression of Rasal1 throughout the cell soma, dendrites and axon which localizes to the neuronal plasma membrane in response to intracellular calcium fluctuation. Pull-down and co-immunoprecipitation demonstrate direct interaction of Rasal1 with PKC, tubulin, and CaMKII. Consequently, Rasal1 is found to stabilize microtubules, through post-translational modification of tubulin, and accordingly inhibit dendritic outgrowth and branching. Through imaging, molecular, and electrophysiological techniques Rasal1 is shown to promote NMDA-mediated synaptic activity and CaMKII phosphorylation. CONCLUSIONS: Rasal1 functions in two separate roles in neuronal development; calcium regulated neurite outgrowth and the promotion of NMDA receptor-mediated postsynaptic events which may be mediated both by interaction with direct binding partners or calcium-dependent regulation of down-stream pathways. Importantly, the outlined molecular mechanisms of Rasal1 may contribute notably to normal neuronal development and synapse formation.

[Study of appropriate cut-off for diagnosis of primary aldosteronism by seated saline suppression test based on liquid chromatography with tandem mass spectrometry].

Objective: To investigate the appropriate cut-off for diagnosis of primary aldosteronism (PA) by seated saline suppression test (SSST) based on liquid chromatography with tandem mass spectrometry (LC-MS/MS). Methods: In this cross-sectional study, patients who underwent SSST for suspected PA in the First Affiliated Hospital of Chongqing Medical University from January 2018 to March 2022 were evaluated. Briefly, 300 patients with PA and 119 with essential hypertension (EH) were included. Plasma aldosterone concentration (PAC) after SSST was determined by LC-MS/MS. Primary aldosteronism confirmatory testing (PACT) score was used as the reference standard for diagnosis of PA, and receiver operating characteristic (ROC) curve was used to explore the cut-off value. Results: The average age of the PA group was (50.8±10.5) years, and males accounted for 53.00% (n=159); the average age of the EH group was (49.4±11.2) years, and males accounted for 26.89% (n=32). The area under the ROC curve of PAC post-SSST was 0.819 (95%CI 0.775-0.862). When 40 pg/ml (110.8 pmol/L) was selected as the appropriate cut-off for diagnosis of PA, the sensitivity was 83.67% (95%CI 78.88%-87.56%) and specificity was 60.50% (95%CI 51.10%-69.21%). Thus, 95.09% (155/163) of patients with unilateral PA could be identified. Conclusion: PAC after SSST determined by LC-MS/MS has high efficacy for diagnosis of PA, and 40 pg/ml is recommended as the appropriate cut-off value.

Bridging the gap: Building environmental, social and governance capabilities in small and medium logistics companies.

Nowadays, the popularity of environmental, social, and governance (ESG) performance measurement has dramatically increased, particularly to listed companies, for supporting various investment decisions. Companies with high ESG scores imply that their ongoing business development is recognised to be economically, socially, and environmentally sustainable. From the current ESG measurement practice, the measurement frameworks are built on rating schemes, such as KLD and ASSET4, so as to derive the ESG scores for listed companies. However, such existing measurement frameworks are difficult to be implemented in small and medium enterprises (SMEs) with unstructured and non-standardised business data, especially in logistics and supply chain management (LSCM) practice. In addition, it is inevitable for listed companies to work with SMEs, for example logistics service providers, but they need a systematic framework to source the responsible SMEs to maintain the ESG performance. To address the above industrial pain-points, this study proposes an ESG development prioritisation and performance measurement framework (ESG-DPPMF) by means of the Bayesian best-worst method enabling the group decision-making capability to prioritise the ESG development areas and formulate the performance measurement scheme. Through consolidating the opinions from logistics practitioners, it is found that fair labour practice, reverse logistics and human right in supply chains are the most essential areas to further enhance ESG capabilities in the logistics industry. In addition, the viability of the ESG performance measurement has been validated, and thus the sustainable and human-centric logistics practice can be developed to achieve business sustainability.

Omega-3 polyunsaturated fatty acids alleviate adenine-induced chronic renal failure via regulating ROS production and TGF-ß/SMAD pathway.

The article "Omega-3 polyunsaturated fatty acids alleviate adenine-induced chronic renal failure via regulating ROS production and TGF-ß/SMAD pathway", by J. Xu, Z.-P. Feng, H.-Y. Peng, P. Fu, published in Eur Rev Med Pharmacol Sci 2018; 22 (15): 5024-5032-DOI: 10.26355/eurrev_201808_15645-PMID: 30070341, has been retracted by the authors due to input errors which occurred in the experimental data. The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/15645.

Investigation on the mechanism of p38MAPK in the failure of autogenous arteriovenous fistula caused by stenosis.

The present study aimed to probe into the mechanism of p38MAPK in the failure of autogenous arteriovenous fistula (AVF) caused by stenosis. A total of 24 patients with maintenance hemodialysis and the autologous AVF as the hemodialysis route were enrolled in the present study. In the experimental group, the internal fistula operation mode was the end-to-side anastomosis, and patients were those who needed autogenous AVF repair due to the venous hyperplasia and stenosis of the internal fistula anastomosis (n = 12). The control group was composed of patients who underwent autogenous AVF surgery for the first time (n = 12). The discarded venous tissues in each group were used for immunohistochemistry and Western blot detection of ASK1, P38, and ATF-2. SPSS 17.0 and GraphPad Prism 5 software were adopted for data analysis. The measurement data were expressed as means ± standard deviations ( x ± s), and P < 0.05 was considered statistically significant. The results of immunohistochemistry staining: the expressions of ASK1, P38, and ATF-2 in the experimental group were significantly higher than those in the control group, and the differences were statistically significant (P < 0.01). The results of Western blot: the expression of P38 in the experimental group was significantly higher than that in the control group, and the difference was statistically significant (P < 0.05). There was no significant difference in the expression of ASK1 and ATF-2 between the experimental group and the control group (P > 0.05). In stenotic AVF, the expressions of ASK1, P38, and ATF-2 all significantly increased, indicating that the p38MAPK signaling pathway might be involved in the formation of venous stenosis in AVF, and the p38MAPK signaling pathway might become a therapeutic target in preventing and treating the vascular stenosis in the fistula.

[Prevalence and clinical characteristics of obstructive sleep apnea in patients with primary hyperaldosteronism].

Objective: To explore the proportion of obstructive sleep apnea (OSA) in primary aldosteronism (PA) in Chinese population and compare the clinical characteristics between PA patients with OSA and those without. Methods: A total of 96 patients diagnosed with PA from September 2015 to November 2018 were recruited in this study. OSA was screened by cardio-respiratory polygraphy. According to the apnea hypopnea index (AHI), the patients were divided into PA with OSA group (AHI ≥5 times) and PA without OSA group (AHI<5 times). Results: Among all patients (96), 69 (71.9%) were with OSA, among them 22 patients (22.9%) were with mild OSA, 17 patients (17.7%) were with moderate OSA and 30 patients (31.3%) were with severe OSA. Compared with the patients without OSA, the patients with OSA were elder, and had higher levels of body mass index (BMI), waist circumference (WC), hip circumference (HC), creatinine (CR) and glycosylated haemoglobin (HbA1c) (P<0.05), but lower concentrations of plasma aldosterone (PAC), supine aldosterone renin concentration ratio(ARR) and the PAC after the diagnosis test (P<0.05). Spearman correlation analyses showed that BMI, WC, HC, CR and HbA1c were positively correlated with AHI (P<0.05), while high-density lipoproteincholesterol (HDL-C), supine-PAC and saline infusion test(SIT)-post PAC were negatively correlated with AHI (P<0.05). Conclusions: The proportion of OSA in PA patients is relatively high (71.9%). Metabolic abnormalities are more common in PA patients with OSA, indicating that screening for OSA should be carried out routinely in PA patients.

[Preliminary study on the application of abdominal aortic balloon occlusion in the treatment of cesarean scar pregnancy].

Objective: To explore the clinical value by analyzing the application of abdominal aortic balloon occlusion in the uterine curettage treatment for patients with cesarean scar pregnancy (CSP). Methods: Totally 42 CSP patients in the first Affiliated Hospital of Zhengzhou University were analyzed retrospectively, 21 cases in the observation group, placing the balloon catheter to the abdominal aorta under the renal artery under the digital substraction angiography(DSA), conducting curettage under hysteroscopy or uterine laparoscopy immediately, and making intermittent blockage in abdominal aorta blood flow during the surgery;21 patients in the control group, conducting uterine artery embolization (UAE) before operation, conducting curettage under hysteroscopy or uterine laparoscopy after 1-3 days. The fluoroscopy time under DSA, body surface radiation dose, intraoperative blood loss, operation time, incidence of postoperative adverse reactions, hospitalization time and follow-up menstruation were comparatively analyzed. Results: All patients operated and retained the uterus successfully. In the control group, all 21 patients had different degrees of fever, pain and other symptoms after UAE. In the observation group and control group, the fluoroscopy time and body surface radiation dose under DSA respectively were (7.4±1.4) s, (5.4±1.1) mGy and (1 142.8±315.5) s, (1 442.0±300.0) mGy (both P<0.01);the average amount of intraoperative blood loss were (22±15), (19±14) ml (P>0.05), the time of uterine curettage were (37±20), (42±19) minutes (P>0.05);hospitalization time were (5.0±0.9), (7.7±1.3) days (P<0.01). The follow-up period was more than 3 months, no adverse reactions were observed in the observation group; 4 cases of menstrual reduction and 1 case of intrauterine adhesions were found in the control group. Conclusion: Abdominal aortic balloon occlusion and UAE could effectively reduce intraoperative bleeding in uterine curettage for patients with CSP; abdominal aortic balloon occlusion has significant reduction of the X-ray dose, shorter hospitalization time, and fewer adverse events comparing to UAE.

Omega-3 polyunsaturated fatty acids alleviate adenine-induced chronic renal failure via regulating ROS production and TGF-ß/SMAD pathway.

OBJECTIVE: To explore the role of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) in adenine-induced rat chronic renal failure and its underlying mechanism. MATERIALS AND METHODS: 30 Sprague Dawley (SD) rats were randomly assigned into three groups, namely sham group, adenine induction group (adenine group) and adenine induction + ω-3 PUFAs treatment group (ω-3 PUFAs group), with 10 rats in each group. Serum and kidney samples were collected after rats were sacrificed. Serum levels of Cr (creatinine) and BUN (urea nitrogen) were detected using commercial kits. HE (hematoxylin and eosin) staining was performed to evaluate the pathological changes of kidneys. Levels of oxidative stress indicators in rat kidney homogenate were detected by relative commercial kits, including SOD (superoxide dismutase), GSH (reduced glutathione), CAT (catalase), and T-AOC (total antioxidant capacity). Reactive oxygen species (ROS) production was also detected by immunofluorescence. Protein expressions of nuclear factor E2 related factor 2 (Nrf2) and transforming growth factor-beta (TGF-ß)/SMAD pathway-related genes were detected by Western blot. RESULTS: Serum levels of Cr and BUN in ω-3 PUFAs group were remarkably decreased compared with those of adenine group. Higher contents of SOD, GSH, CAT and T-AOC were observed in ω-3 PUFAs group compared with those of adenine group. Besides, MAD content and ROS production were lower in ω-3 PUFAs group than those of adenine group. Pathological changes of kidneys were alleviated after ω-3 PUFAs treatment. Western blot results demonstrated that ω-3 PUFAs treatment remarkably upregulates Nrf2, HO-1, NQO1, but downregulates relative genes in TGF-ß/SMAD pathway. CONCLUSIONS: ω-3 PUFAs alleviated adenine-induced chronic renal failure through enhancing antioxidant stress and inhibiting inflammatory response via regulating Nrf2 and TGF-ß/SMAD pathway.

Carvacrol promotes neuroprotection in the mouse hemiparkinsonian model.

Carvacrol is a monoterpene that has been linked to neuroprotection in several animal models of neurodegeneration, including ischemia, epilepsy and traumatic neuronal injury. In this study, we investigated the effects of carvacrol (i.p.) upon the neurodegeneration induced by 6-hydroxy-dopamine unilateral intrastriatal injections in mice. We have also used the cylinder test to assess the behavioral effects of carvacrol in that model of Parkinson's disease, and immunoblots to evaluate the levels of caspase-3 and TRPM7, one of major targets of carvacrol. Behavioral testing revealed that carvacrol largely reduced the asymmetrical use of the forelimbs induced by unilateral 6-hydroxy-dopamine. Carvacrol dramatically reduced the loss of tyrosine hydroxylase immunostaining both in the substantia nigra and in the striatum that are typical of the model. Immunoblots for tyrosine hydroxylase confirmed this effect. Caspase-3 levels were very high after toxin injections, but carvacrol appeared to reduce them to control levels. Finally, TRPM7, observed by immunoblots, increased after 6-hydroxy-dopamine, suggesting the involvement of this cation channel in the ensuing neurodegenerative process. The present data suggest that carvacrol promotes a marked neuroprotection in the 6-hydroxy-dopamine model of Parkinson's disease, possibly by its non-specific blocking effect upon TRPM7 channels.

High sensitivity of spontaneous spike frequency to sodium leak current in a Lymnaea pacemaker neuron.

The spontaneous rhythmic firing of action potentials in pacemaker neurons depends on the biophysical properties of voltage-gated ion channels and background leak currents. The background leak current includes a large K+ and a small Na+ component. We previously reported that a Na+ -leak current via U-type channels is required to generate spontaneous action potential firing in the identified respiratory pacemaker neuron, RPeD1, in the freshwater pond snail Lymnaea stagnalis. We further investigated the functional significance of the background Na+ current in rhythmic spiking of RPeD1 neurons. Whole-cell patch-clamp recording and computational modeling approaches were carried out in isolated RPeD1 neurons. The whole-cell current of the major ion channel components in RPeD1 neurons were characterized, and a conductance-based computational model of the rhythmic pacemaker activity was simulated with the experimental measurements. We found that the spiking rate is more sensitive to changes in the Na+ leak current as compared to the K+ leak current, suggesting a robust function of Na+ leak current in regulating spontaneous neuronal firing activity. Our study provides new insight into our current understanding of the role of Na+ leak current in intrinsic properties of pacemaker neurons.

Involvement of AP-1 in p38MAPK signaling pathway in osteoblast apoptosis induced by high glucose.

We investigated the effect of p38MAPK/AP-1 (activator protein-1) signaling on the apoptosis of osteoblasts induced by high glucose. A lentivirus vector of small hairpin RNA (shRNA) targeting p38MAPK was constructed in vitro. Osteoblasts MC3T3-E1 cultured in vitro were treated with vehicle, high glucose, p38MAPK-shRNA transfection, p38MAPK inhibitor, and unrelated shRNA transfection. Apoptosis, protein levels of p38MAPK, and activities of AP-1 in MC3T3-E1 osteoblasts were measured using TUNEL and flow cytometry, Western blot analysis, and an electrophoretic mobility shift assay. Compared with the vehicle group, high glucose induced apoptosis of MC3T3-E1 osteoblasts and activated p38MAPK and AP-1. p38MAPK-shRNA transfection blocked the effect of high glucose stimulation, and the p38MAPK inhibitor showed similar effects as those observed in p38MAPK transfection. Unrelated shRNA had no effect on these changes in MC3T3-E1 osteoblasts induced by high glucose. Therefore, our results suggest that p38MAPK-shRNA reduce apoptosis of MC3T3-E1 osteoblasts induced by high glucose by inhibiting the p38MAPK-AP-1 signaling pathway.

Expression, phosphorylation, and glycosylation of CNS proteins in aversive operant conditioning associated memory in Lymnaea stagnalis.

Long-term memory formation requires "de novo" expression and post-translational modification of many proteins. Understanding the temporal and spatial regulatory pattern of these proteins is fundamental to decoding the molecular basis of learning and memory. We characterized changes in expression, phosphorylation, and glycosylation of CNS proteins after operant conditioning in pond snail Lymnaea stagnalis. The phosphorylation and the glycosylation levels of proteins, measured by the ratio of Pro-Q Diamond (phosphoproteins) or Pro-Q Emerald (glycoproteins) vs. SYPRO-Ruby (total proteins) signals, increased during memory formation. Proteins whose modulation of phosphorylation might be involved in learning and memory were identified by mass spectrometry (MS) and are associated with cytoskeleton, glutamine cycle, energy metabolism, G-protein signaling, neurotransmitter release regulation, iron transport, protein synthesis, and cell division. Phosphorylation of actin increased during memory formation. To identify proteins whose expression levels changed in long-term memory formation we used two-dimensional difference gel electrophoresis followed by MS. The up-regulated proteins are mostly associated with lipoprotein and cholesterol metabolism, protein synthesis and degradation, cytoskeleton, nucleic acid synthesis, and energy supply. The down-regulated proteins are enzymes of aspartic acid metabolism involved in regulation of protein synthesis. Our proteomic analyses have revealed a number of candidate proteins associated with memory formation. These findings provide new directions for further investigation into the signaling networks required for memory formation and consolidation.

Transcriptome analysis of the central nervous system of the mollusc Lymnaea stagnalis.

BACKGROUND: The freshwater snail Lymnaea stagnalis (L. stagnalis) has served as a successful model for studies in the field of Neuroscience. However, a serious drawback in the molecular analysis of the nervous system of L. stagnalis has been the lack of large-scale genomic or neuronal transcriptome information, thereby limiting the use of this unique model. RESULTS: In this study, we report 7,712 distinct EST sequences (median length: 847 nucleotides) of a normalized L. stagnalis central nervous system (CNS) cDNA library, resulting in the largest collection of L. stagnalis neuronal transcriptome data currently available. Approximately 42% of the cDNAs can be translated into more than 100 consecutive amino acids, indicating the high quality of the library. The annotated sequences contribute 12% of the predicted transcriptome size of 20,000. Surprisingly, approximately 37% of the L. stagnalis sequences only have a tBLASTx hit in the EST library of another snail species Aplysia californica (A. californica) even using a low stringency e-value cutoff at 0.01. Using the same cutoff, approximately 67% of the cDNAs have a BLAST hit in the NCBI non-redundant protein and nucleotide sequence databases (nr and nt), suggesting that one third of the sequences may be unique to L. stagnalis. Finally, using the same cutoff (0.01), more than half of the cDNA sequences (54%) do not have a hit in nematode, fruitfly or human genome data, suggesting that the L. stagnalis transcriptome is significantly different from these species as well. The cDNA sequences are enriched in the following gene ontology functional categories: protein binding, hydrolase, transferase, and catalytic enzymes. CONCLUSION: This study provides novel molecular insights into the transcriptome of an important molluscan model organism. Our findings will contribute to functional analyses in neurobiology, and comparative evolutionary biology. The L. stagnalis CNS EST database is available at http://www.Lymnaea.org/.

Neural phosphoproteomics of a chronic hypoxia model--Lymnaea stagnalis.

Chronic hypoxia is a common clinical event that induces adaptive responses and can result in behavioral deterioration. The reduction of metabolic rate during hypoxia may limit overall protein phosphorylation owing to the lack of high energy phosphate. However, the hypoxia-induced regulation of phosphoproteins is poorly understood. Here, we characterized the CNS phosphoproteome of Lymnaea stagnalis, a freshwater snail that has been used as a model to study chronic hypoxia-induced neural depression. After hypoxia treatment for 4 days, the motor behavior of the snail was suppressed. Electrophysiological measurements from Pedal A (PeA) interneurons showed that hypoxia increased the frequency of spontaneous postsynaptic excitatory potentials (sEPSPs), but reduced the firing frequency, the amplitude, and the half-width duration (APD(50)) of spontaneous action potentials. Imaging with a fluorescent phosphate label, Pro-Q Diamond, revealed that the neuronal phosphoprotein level was reduced after the hypoxia treatment. The hypoxia-induced changes in the phosphoproteome of the central ganglia were quantified using one-dimensional gel-electrophoresis by comparing the fluorescence intensity ratio of phospholabeled phosphoproteins versus total proteins between the hypoxia and control groups. We analyzed 16 protein bands: eight showed decreased phosphorylation levels after hypoxia treatment, and eight did not change. Using mass spectrometry analysis and protein database matching we found three phosphoproteins that may be associated with chronic hypoxia-induced neuronal adaptive response of the snail. This is the first proteomic screening for neural phosphoproteins in chronic hypoxia.

Chronic hypoxia-induced alteration of presynaptic protein profiles and neurobehavioral dysfunction are averted by supplemental oxygen in Lymnaea stagnalis.

Chronic hypoxia causes neural dysfunction. Oxygen (O(2)) supplements have been commonly used to increase the O(2) supply, yet the therapeutic benefit of this treatment remains controversial due to a lack of cellular and molecular evidence. In this study, we examined the effects of short-burst O(2) supplementation on neural behavior and presynaptic protein expression profiles in a simple chronic hypoxia model of snail Lymnaea stagnalis. We reported that hypoxia delayed the animal response to light stimuli, suppressed locomotory activity, induced expression of stress-response proteins, hypoxia inducible factor-1alpha (HIF-1alpha) and heat shock protein 70 (HSP70), repressed syntaxin-1 (a membrane-bound presynaptic protein) and elevated vesicle-associated membrane protein-1 (VAMP-1) (a vesicle-bound presynaptic protein) level. O(2) supplements relieved suppression of neural behaviors, and corrected hypoxia-induced protein alterations in a dose-dependent manner. The effectiveness of supplemental O(2) was further evaluated by determining time courses for recovery of neural behaviors and expression of stress response proteins and presynaptic proteins after relief from hypoxia conditions. Our findings suggest that O(2) supplement improves hypoxia-induced adverse alterations of presynaptic protein expression and neurobehaviors, however, the optimal level of O(2) required for improvement is protein specific and system specific.

Kir6.2-containing ATP-sensitive potassium channels protect cortical neurons from ischemic/anoxic injury in vitro and in vivo.

ATP-sensitive potassium (K(ATP)) channels are weak inward rectifiers that appear to play an important role in protecting neurons against ischemic damage. Cerebral stroke is a major health issue, and vulnerability to stroke damage is regional within the brain. Thus, we set out to determine whether K(ATP) channels protect cortical neurons against ischemic insults. Experiments were performed using Kir6.2(-/-) K(ATP) channel knockout and Kir6.2(+/+) wildtype mice. We compared results obtained in Kir6.2(-/-) and wildtype mice to evaluate the protective role of K(ATP) channels against focal ischemia in vivo, and, using cortical slices, against anoxic stress in vitro. Immunohistochemistry confirmed the presence of K(ATP) channels in the cortex of wildtype, but not Kir6.2(-/-), mice. Results from in vivo and in vitro experimental models indicate that Kir6.2-containing K(ATP) channels in the cortex provide protection from neuronal death. Briefly, in vivo focal ischemia (15 min) induced severe neurological deficits and large cortical infarcts in Kir6.2(-/-) mice, but not in wildtype mice. Imaging analyses of cortical slices exposed briefly to oxygen and glucose deprivation (OGD) revealed a substantial number of damaged cells (propidium iodide-labeled) in the Kir6.2(-/-) OGD group, but few degenerating neurons in the wildtype OGD group, or in the wildtype and Kir6.2(-/-) control groups. Slices from the three control groups had far more surviving cells (anti-NeuN antibody-labeled) than slices from the Kir6.2(-/-) OGD group. These findings suggest that stimulation of endogenous cortical K(ATP) channels may provide a useful strategy for limiting the damage that results from cerebral ischemic stroke.