Potential effects of Cu2+ stress on nitrogen removal performance, microbial characteristics, and metabolism pathways of biofilm reactor.

Sequencing batch biofilm reactors (SBBR) were utilized to investigate the impact of Cu2+ on nitrogen (N) removal and microbial characteristics. The result indicated that the low concentration of Cu2+ (0.5 mg L-1) facilitated the removal of ammonia nitrogen (NH4+-N), total nitrogen (TN), nitrate nitrogen (NO3--N), and chemical oxygen demand (COD). In comparison to the average effluent concentration of the control group, the average effluent concentrations of NH4+-N, NO3--N, COD, and TN were found to decrease by 40.53%, 17.02%, 10.73%, and 15.86%, respectively. Conversely, the high concentration of Cu2+ (5 mg L-1) resulted in an increase of 94.27%, 55.47%, 22.22%, and 14.23% in the aforementioned parameters, compared to the control group. Low concentrations of Cu2+ increased the abundance of nitrifying bacteria (Rhodanobacter, unclassified-o-Sacharimonadales), denitrifying bacteria (Thermomonas, Comamonas), denitrification-associated genes (hao, nosZ, norC, nffA, nirB, nick, and nifD), and heavy-metal-resistant genes related to Cu2+ (pcoB, cutM, cutC, pcoA, copZ) to promote nitrification and denitrification. Conversely, high concentration Cu2+ hindered the interspecies relationship among denitrifying bacteria genera, nitrifying bacteria genera, and other genera, reducing denitrification and nitrification efficiency. Cu2+ involved in the N and tricarboxylic acid (TCA) cycles, as evidenced by changes in the abundance of key enzymes, such as (EC:1.7.99.1), (EC:1.7.2.4), and (EC:1.1.1.42), which initially increased and then decreased with varying concentrations of Cu2+. Conversely, the abundance of EC1.7.2.1, associated with the accumulation of nitrite nitrogen (NO2--N), gradually declined. These findings provided insights into the impact of Cu2+ on biological N removal.

Unraveling the effects and mechanisms of microplastics on anaerobic fermentation: Exploring microbial communities and metabolic pathways.

To investigate the effects of microplastics (MPs) on hydrolysis, acidification and microbial characteristics during waste activated sludge (WAS) anaerobic fermentation process, five different kinds of MPs were added into the WAS fermentation system and results indicated that, compared to the control group, the addition of polyvinyl chloride (PVC)-MPs exhibited the least inhibition on volatile fatty acids (VFAs), reducing them by 13.49 %. Conversely, polyethylene (PE)-MPs resulted in the greatest inhibition, with a reduction of 29.57 %. MPs, while accelerated the dissolution of WAS that evidenced by an increase of lactate dehydrogenase (LDH) release, concurrently inhibited the activities of relevant hydrolytic enzymes (α-Glucosidase, protease). For microbial mechanisms, MPs addition affected the proliferation of key microorganisms (norank_f_Bacteroidetes_vadinHA17, Ottowia, and Propioniclava) and reduced the abundance of genes associated with hydrolysis and acidification (pfkb, gpmI, ilvE, and aces). Additionally, MPs decreased the levels of key hydrolytic and acidogenic enzymes to inhibit hydrolysis and acidification processes. This research provides a basis for understanding and unveils impact mechanisms of the impact of MPs on sludge anaerobic fermentation.

Clinical features, laboratory findings and treatment of juxtaglomerular cell tumors: a systemic review.

Juxtaglomerular cell tumors (JGCTs) or reninoma are rare kidney tumors leading to secondary hypertension, and the non-specific clinical manifestations bring about challenges to the diagnosis. This study is to summarize the clinical features, laboratory findings, and treatment of JGCTs. The PubMed, EMBASE database, and manual search were utilized to find all cases, and 158 reports containing 261 patients were identified. Data on patients' demographics, clinical features, diagnostic methods, and treatment options were collected and analyzed. JGCTs occurred predominantly in female patients (female to male ratio, 2.1:1). The median age of patients was 25 years (IQR:18-34 years). Hypertension (97.24%) was the cardinal manifestation. Hypokalemia was reported in 78.71% (159/202) of subjects, and normal serum potassium accounted for 20.79% (42/202). In cases with assessed plasma renin activity (PRA) levels, the median PRA was 7.89 times the upper limit of normal (IQR:3.58-14.41), and 3.82% (5/131) of cases in the normal range. Tumors were detected in 97.8% (175/179) computed tomography (CT), 94.7% (72/76) magnetic resonance imaging (MRI), and 81.5% (110/135) ultrasound, respectively. For 250/261 patients undergoing surgical procedures, 89.14% (197/221), 94.94% (150/158), and 100% (131/131) of patients were restored to normal blood pressure, PRA, and serum potassium, respectively. JGCTs are commonly associated with hypertension, hypokalemia, and hyperreninemia, whereas patients with normotension, normokalemia, and PRA should be systematically pursued after drug-elution lasting for 2 weeks. CT and MRI are more sensitive imaging diagnostic methods. The blood pressure and biochemical parameters of most patients returned to normal after surgery.

Decoupled electrolysis for hydrogen production and hydrazine oxidation via high-capacity and stable pre-protonated vanadium hexacyanoferrate.

Decoupled electrolysis for hydrogen production with the aid of a redox mediator enables two half-reactions operating at different rates, time, and spaces, which offers great flexibility in operation. Herein, a pre-protonated vanadium hexacyanoferrate (p-VHCF) redox mediator is synthesized. It offers a high reversible specific capacity up to 128 mAh g-1 and long cycling performance of 6000 cycles with capacity retention about 100% at a current density of 10 A g-1 due to the enhanced hydrogen bonding network. By using this mediator, a membrane-free water electrolytic cell is built to achieve decoupled hydrogen and oxygen production. More importantly, a decoupled electrolysis system for hydrogen production and hydrazine oxidation is constructed, which realizes not only separate hydrogen generation but electricity generation through the p-VHCF-N2H4 liquid battery. Therefore, this work enables the flexible energy conversion and storage with hydrogen production driven by solar cell at day-time and electricity output at night-time.

A New Scoring System Predicting Side-Branch Occlusion in Patients Undergoing Left Main Bifurcation Intervention: The LM V-RESOLVE Score.

BACKGROUND: The risk of side-branch (SB) occlusion is pivotal for decision making of stenting strategies during unprotected left main (LM) bifurcation percutaneous coronary intervention (PCI). Accordingly, this study aimed to develop a scoring system for predicting SB occlusion during unprotected LM bifurcation PCI. METHODS: A total of 855 consecutive patients undergoing unprotected LM bifurcation PCI with provisional strategy at Fuwai Hospital from January 2014 to December 2016 were recruited. A prediction model was selected by means of all-subsets logistic regression, and a multivariable risk score (Left Main Visual Estimation for Risk Prediction of Side Branch Occlusion in Coronary Bifurcation Intervention [LM V-RESOLVE]) was then established with incremental weights attributed to each component variable based on its estimate coefficients. SB occlusion was defined as any decrease in Thrombolysis in Myocardial Infarction (TIMI) flow grade or absence of flow in SB after main vessel (MV) stenting. RESULTS: SB occlusion occurred in 19 LM bifurcation lesions (2.22%). In multivariable model, 3 variables, including MV/SB diameter ratio, MV plaque ipsilateral to SB, and baseline diameter stenosis of SB, were independent predictors for SB occlusion (model C-statistic 0.829, 95% confidence interval [CI] 0.735-0.923, with good calibration). The risk score had a C-statistics of 0.830 (95% CI 0.738-0.923) with good calibration. Satisfactory discriminative ability of the risk score was also preserved in external validation (C-statistic 0.794, 95% CI 0.691-0.896). CONCLUSIONS: The LM bifurcation-specific novel scoring system, LM V-RESOLVE, based on 3 simple baseline angiographic findings, could help to rapidly discriminate lesions at risk of SB occlusion during LM bifurcation PCI.

Evaluation of Two Chemoenzymatic Glycan Remodeling Approaches to Generate Site-Specific Antibody-Drug Conjugates.

Fc-glycosite-specific antibody-drug conjugation represents a promising direction for the preparation of site-specific antibody-drug conjugates (ADCs). In the present research, we conducted a systemic evaluation of two endoglycosidase-catalyzed chemoenzymatic glycoengineering technologies to prepare glycosite-specific ADCs. In the first two-step approach, the antibody was deglycosylated and then reglycosylated with a modified intact N-glycan oxazoline. In the second one-pot approach, antibodies were deglycosylated and simultaneously glycosylated with a functionalized disaccharide oxazoline. For the comprehensive evaluation, we first optimized and scaled-up the preparation of azido glycan oxazolines. Afterwards, we proved that the one-pot glycan-remodeling approach was efficient for all IgG subclasses. Subsequently, we assembled respective ADCS using two technology routes, with two different linker-payloads combinations, and performed systemic in vitro and in vivo evaluations. All the prepared ADCs achieved high homogeneity and illustrated excellent stability in buffers with minimum aggregates, and exceptional stability in rat serum. All ADCs displayed a potent killing of BT-474 breast cancer cells. Moving to the mouse study, the ADCs prepared from two technology routes displayed potent and similar efficacy in a BT-474 xenograft model, which was comparable to an FDA-approved ADC generated from random conjugation. These ADCs also demonstrated excellent safety and did not cause body weight loss at the tested dosages.

A retrospective three-dimensional study of the mandibular growth during preoperative orthodontics in late adolescent patients with skeletal Class III malocclusion.

INTRODUCTION: The mandible's ongoing development presents a contraindication for combined orthodontic-orthognathic treatment. The aim of this study was to evaluate the mandibular stability before and after preoperative orthodontic treatment in late adolescent patients with skeletal Class III malocclusion and to investigate the most appropriate time to start preoperative orthodontic treatment. MATERIAL AND METHODS: The study population consisted of 58 adolescents, aged between 15 and 21 years, with skeletal Class III malocclusion; the adolescents underwent CT scans at the beginning (T1) and the end (T2) of preoperative orthodontic treatment. The CT data were analyzed using ITK-SNAP and 3D Slicer software, and the effects of age and gender on mandibular development were investigated. RESULTS: In these 58 patients, there were no significant local bone alterations in the condyle and anterior chin point between T1 and T2 and no significant changes in the mandibular branch height, mandibular body length, condylar distance, and mandibular angle distance (p>0.05). The mandibular growth at the angel of mandible was statistically significant (p<0.05), but it was not clinically significant because the mean value of the growth was small (right: 0.416±0.986 mm, left: 0.328±0.886 mm). No effect of age and gender on mandibular development was observed. CONCLUSION: The mandibular morphology was stable during preoperative orthodontic treatment in patients at the late adolescent stage. This study provides evidence for the possibility of early implementation of preoperative orthodontics.

Decoupled alkaline water electrolysis by a K0.5MnO2-Ti mediator via K-ion insertion/extraction.

Conventional one-step water electrolyzers generate H2 accompanied by O2 evolution, and may cause gas mixing and high cell voltage inputs. Herein, using the potassium ion battery material of K0.5MnO2-Ti as a mediator, we effectively decoupled the H2 and O2 evolution of alkaline water electrolysis temporally, thereby achieving a membrane-free pathway for H2 production. As a mediator electrode for charge storage, the K0.5MnO2-Ti exhibited a stable capacity of 100 mA h g-1 at 0.1 A g-1 owing to the reversible K-ion insertion/extraction mechanism. The decoupled water electrolysis device exhibited the step voltages for hydrogen and oxygen production of 1.02 and 0.57 V at 5 mA, respectively. A nearly unity Faradaic efficiency and sustained production of pure H2 has been demonstrated at a constant current density. We anticipate that this mediator demonstrated here may provide a route for the practical application of the decoupling strategy.

LC-MS/MS-PRM Quantification of IgG Glycoforms Using Stable Isotope Labeled IgG1 Fc Glycopeptide Standard.

Targeted quantification of proteins is a standard methodology with broad utility, but targeted quantification of glycoproteins has not reached its full potential. The lack of optimized workflows and isotopically labeled standards limits the acceptance of glycoproteomics quantification. In this work, we introduce an efficient and streamlined chemoenzymatic synthesis of a library of isotopically labeled glycopeptides of IgG1 which we use for quantification in an energy optimized LC-MS/MS-PRM workflow. Incorporation of the stable isotope labeled N-acetylglucosamine enables an efficient monitoring of all major fragment ions of the glycopeptides generated under the soft higher-energy C-trap dissociation (HCD) conditions, which reduces the coefficients of variability (CVs) of the quantification to 0.7-2.8%. Our results document, for the first time, that the workflow using a combination of stable isotope labeled standards with intrascan normalization enables quantification of the glycopeptides by an electron transfer dissociation (ETD) workflow, as well as the HCD workflow, with the highest sensitivity compared to traditional workflows. This was exemplified by a rapid quantification (13 min) of IgG1 Fc glycoforms from COVID-19 patients.

LC-MS/MS-PRM Quantification of IgG glycoforms using stable isotope labeled IgG1 Fc glycopeptide standard.

Targeted quantification of proteins is a standard methodology with broad utility, but targeted quantification of glycoproteins has not reached its full potential. The lack of optimized workflows and isotopically labeled standards limits the acceptance of glycoproteomics quantification. In this paper, we introduce an efficient and streamlined chemoenzymatic synthesis of a library of isotopically labeled glycopeptides of IgG1 which we use for quantification in an energy optimized LC-MS/MS-PRM workflow. Incorporation of the stable isotope labeled N-acetylglucosamine enables an efficient monitoring of all major fragment ions of the glycopeptides generated under the soft collision induced dissociation (CID) conditions which reduces the CVs of the quantification to 0.7-2.8%. Our results document, for the first time, that the workflow using a combination of stable isotope labeled standards with intra-scan normalization enables quantification of the glycopeptides by an electron transfer dissociation (ETD) workflow as well as the CID workflow with the highest sensitivity compared to traditional workflows., This was exemplified by a rapid quantification (13-minute) of IgG1 Fc glycoforms from COVID-19 patients.

Structured sampling of olfactory input by the fly mushroom body.

Associative memory formation and recall in the fruit fly Drosophila melanogaster is subserved by the mushroom body (MB). Upon arrival in the MB, sensory information undergoes a profound transformation from broadly tuned and stereotyped odorant responses in the olfactory projection neuron (PN) layer to narrowly tuned and nonstereotyped responses in the Kenyon cells (KCs). Theory and experiment suggest that this transformation is implemented by random connectivity between KCs and PNs. However, this hypothesis has been challenging to test, given the difficulty of mapping synaptic connections between large numbers of brain-spanning neurons. Here, we used a recent whole-brain electron microscopy volume of the adult fruit fly to map PN-to-KC connectivity at synaptic resolution. The PN-KC connectome revealed unexpected structure, with preponderantly food-responsive PN types converging at above-chance levels on downstream KCs. Axons of the overconvergent PN types tended to arborize near one another in the MB main calyx, making local KC dendrites more likely to receive input from those types. Overconvergent PN types preferentially co-arborize and connect with dendrites of αß and α'ß' KC subtypes. Computational simulation of the observed network showed degraded discrimination performance compared with a random network, except when all signal flowed through the overconvergent, primarily food-responsive PN types. Additional theory and experiment will be needed to fully characterize the impact of the observed non-random network structure on associative memory formation and recall.

Changes in T Lymphocyte Subsets in Different Tumors Before and After Radiotherapy: A Meta-analysis.

Purpose: Radiation therapy (RT) induces an immune response, but the relationship of this response with tumor type is not fully understood. This meta-analysis further elucidated this relationship by analyzing the changes in T lymphocyte subsets in different tumors before and after radiotherapy. Methods: We searched English-language electronic databases including PubMed, EMBASE, and the Cochrane Library to collect studies on the changes in peripheral blood CD3+ T lymphocytes, CD4+ T lymphocytes, and CD8+ T lymphocytes before and after radiotherapy in tumor patients from January 2015 to April 2021. The quality of the included literature was evaluated using the NOS scale provided by the Cochrane Collaboration, and statistical software RevMan 5.4 was used to analyze the included literature. P<0.05 was considered to indicate statistical significance. Results: A total of 19 studies in 16 articles involving 877 tumor patients were included. All data were collected within 1 month before or after radiotherapy. Meta-analysis showed that numbers of CD3+ T lymphocytes (SMD: -0.40; 95% CI [-0.75, -0.04]; p = 0.03) and CD4+ T lymphocytes (SMD: -0.43; 95% CI: [-0.85, -0.02]; p = 0.04) were significantly reduced after radiotherapy compared with before treatment, but there was no statistically significant difference for CD8+ T lymphocytes (SMD: 0.33; 95% CI: [-0.88, 0.74]; p = 0.12). Subgroup analysis showed that peripheral blood T lymphocytes decreased in head and neck cancer. However, in prostate cancer and breast cancer, there was no significant change in peripheral blood. 1 month after radiotherapy, it has a potential proliferation and activation effect on lymphocytes in esophageal cancer and lung cancer. The results showed that CD8+T lymphocytes increased in peripheral blood after SBRT. Radiotherapy alone reduced CD3+ T lymphocyte numbers. Conclusions: Within 1 month of radiotherapy, patients have obvious immunological changes, which can cause apoptosis and reduction of T lymphocytes, and affect the balance of peripheral blood immune cells. The degree of immune response induced by radiotherapy differed between tumor types.

Circulating miRNAs in Serum as Biomarkers for Early Diagnosis of Non-small Cell Lung Cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) accounts for about 85% of lung cancers. This study aimed to discover the potential miRNA biomarkers for early detection of NSCLC. METHODS: Total circulating miRNAs were extracted from six patients and six volunteers and run on the miRNA chip. The differentially expressed miRNAs acquired by data mining were intersected with chip results, and qRT-PCR were carried out. Then the differentially miRNAs were validated by using a validation cohort (120 participants). ROC curves were established to evaluate the diagnostic efficacy of the differentially circulating miRNAs. The target genes of the differential miRNAs were identified using the miRTarBase database, and follow-up GO and KEGG enrichment analysis were conducted. RESULTS: We identified 577 miRNA which screened according to the criteria (fold change > 2 and p value < 0.05). Among them, seven circulating miRNAs passed additional filtering based on data mining. These miRNAs were further validated in the training and validation cohort. miR-492, miR-590-3p, and miR-631 were differentially expressed in the patients' serum, and the area under the ROC curve (AUC) values of these miRNAs were 0.789, 0.792, and 0.711, respectively. When using them as a combination to discriminate healthy volunteers from patients, the AUC reached 0.828 (95% CI, 0.750-0.905, p = 0.000) with a sensitivity of 86.7% and specificity of 71.7%. The follow-up enrichment analysis showed that target genes of three miRNA were associated with tumorigenesis and progression, such as cell cycle and P53 signaling pathway. CONCLUSIONS: The combination of miR-492, miR-590-3p, and miR-631 can be utilized to distinguish healthy individuals and early-stage NSCLC patients. IMPACT: The combination of miR-492, miR-590-3p, and miR-631 might be a promising serum biomarker in patients for the early diagnosis of NSCLC.

Atypical Myosin Tunes Dendrite Arbor Subdivision.

Dendrite arbor pattern determines the functional characteristics of a neuron. It is founded on primary branch structure, defined through cell intrinsic and transcription-factor-encoded mechanisms. Developing arbors have extensive acentrosomal microtubule dynamics, and here, we report an unexpected role for the atypical actin motor Myo6 in creating primary branch structure by specifying the position, polarity, and targeting of these events. We carried out in vivo time-lapse imaging of Drosophila adult sensory neuron differentiation, integrating machine-learning-based quantification of arbor patterning with molecular-level tracking of cytoskeletal remodeling. This revealed that Myo6 and the transcription factor Knot regulate transient surges of microtubule polymerization at dendrite tips; they drive retrograde extension of an actin filament array that specifies anterograde microtubule polymerization and guides these microtubules to subdivide the tip into multiple branches. Primary branches delineate functional compartments; this tunable branching mechanism is key to define and diversify dendrite arbor compartmentalization.

Mussel-Inspired Surface Functionalization of AEM for Simultaneously Improved Monovalent Anion Selectivity and Antibacterial Property.

A facile membrane surface modification process for improving permselectivity and antimicrobial property was proposed. A polydopamine (PDA) coating was firstly fabricated on pristine anion exchange membrane (AEM), followed by in situ reduction of Ag without adding any extra reductant. Finally, 2,5-diaminobenzene sulfonic acid (DSA) was grafted onto PDA layer via Michael addition reaction. The as-prepared AEM exhibited improved permselectivity (from 0.60 to 1.43) and effective inhibition of bacterial growth. In addition, the result of the long-term (90-h continuous electrodialysis) test expressed the excellent durability of the modified layer on membrane surface, because the concentration of Cl- and SO4²- in diluted chamber fluctuated ~0.024 and 0.030 mol·L-1 with no distinct decline. The method described in this work makes the full use of multifunctional PDA layer (polymer-like coating, in situ reduction and post-organic reaction), and a rational design of functional AEM was established for better practical application.

Framework for fusing traffic information from social and physical transportation data.

Tremendous volumes of messages on social media platforms provide supplementary traffic information and encapsulate crowd wisdom for solving transportation problems. However, social media messages manifested in human languages are usually characterized with redundant, fuzzy and subjective features. Here, we develop a data fusion framework to identify social media messages reporting non-recurring traffic events by connecting the traffic events with traffic states inferred from taxi global positioning system (GPS) data. Temporal-spatial information of traffic anomalies caused by the traffic events are then retrieved from anomalous traffic states. The proposed framework successfully identified accidental traffic events with various scales and exhibited strong performance in event descriptions. Even though social media messages are generally posted after the occurrence of anomalous traffic states, resourceful event descriptions in the messages are helpful in explaining traffic anomalies and for deploying suitable countermeasures.

A Complete Electron Microscopy Volume of the Brain of Adult Drosophila melanogaster.

Drosophila melanogaster has a rich repertoire of innate and learned behaviors. Its 100,000-neuron brain is a large but tractable target for comprehensive neural circuit mapping. Only electron microscopy (EM) enables complete, unbiased mapping of synaptic connectivity; however, the fly brain is too large for conventional EM. We developed a custom high-throughput EM platform and imaged the entire brain of an adult female fly at synaptic resolution. To validate the dataset, we traced brain-spanning circuitry involving the mushroom body (MB), which has been extensively studied for its role in learning. All inputs to Kenyon cells (KCs), the intrinsic neurons of the MB, were mapped, revealing a previously unknown cell type, postsynaptic partners of KC dendrites, and unexpected clustering of olfactory projection neurons. These reconstructions show that this freely available EM volume supports mapping of brain-spanning circuits, which will significantly accelerate Drosophila neuroscience. VIDEO ABSTRACT.

Minimization of the perianal infection rate of hematological malignancies with agranulocytosis by quality control circle activity and patient-hospital-student win-win concept.

Objective The agranulocytosis-associated perianal infection (PI) rate ranges from 60% to 100% among patients with hematopoietic malignancies. In this study, we assessed the efficacy of a quality control circle (QCC) to minimize the PI rate. Methods Among 274 patients with severe immunodeficiency (agranulocytosis of ≥2 weeks) in our bone marrow transplantation center, the PI rate was 17.20%. A QCC was established following the 10 steps of the plan-do-check-act (PDCA) model; this was scientifically supported by culturing the bacterial colony from patients' perianal skin to determine the sanitization effect and interval time. Because a warm aqueous solution of potassium permanganate is recommended for sanitization, the bacterial colony culture was also used to determine the proper drug concentration, water temperature, and soaking time. All procedures were standardized. Patients, hospital staff, and medical students were enrolled into the QCC team based on the patient-hospital-student (PHS) win-win concept. Results After establishment of the PDCA model, the PI rate among 253 patients decreased from 17.20% to 5.93% and remained at 5.25% during the following year. The medical expenses and length of hospital stay consequently decreased. Conclusion The QCC and PHS win-win concept can reduce the PI rate and promote medical quality.

Egr-1 transactivates Bim gene expression to promote neuronal apoptosis.

The proapoptotic BH3-only protein Bim is a crucial regulator of neuronal apoptosis. Previous studies have indicated the involvement of the c-Jun, FOXO1/3a, and B/C-Myb transcription factors in the regulation of Bim during neuronal apoptosis. However, the mechanism underlying the transcriptional regulation of Bim in activity deprivation-induced neuronal apoptosis has remained unclear. The present study demonstrates that early growth response 1 (Egr-1), rather than c-Jun, FOXO1/3a, or B/C-Myb, directly transactivates Bim gene expression to mediate apoptosis of rat cerebellar granule neurons. We showed that Egr-1 was sufficient and necessary for neuronal apoptosis. Suppression of Egr-1 activity using dominant-negative mutant or knockdown of Egr-1 using small interfering RNAs led to a decrease in Bim expression, whereas overexpression of Egr-1 resulted in induction of Bim. Deletion and site-directed mutagenesis of the Bim promoter revealed that Bim transcriptional activation depends primarily on a putative Egr-binding sequence between nucleotides -56 and -47 upstream of the start site. We also showed that Egr-1 binding to this sequence increased in response to activity deprivation in vitro and in vivo. Moreover, inhibition of Egr-1 binding to the Bim promoter, by mithramycin A and chromomycin A3, reduced the activity deprivation-induced increases in Bim promoter activity and mRNA and protein levels and protected neurons from apoptosis, further supporting the Egr-1-mediated transactivation of Bim. Additionally, Bim overcame the Egr-1 knockdown-mediated inhibition of apoptosis, whereas Bim knockdown impaired the increase in apoptosis induced by Egr-1. These findings establish Bim as an Egr-1 target gene in neurons, uncovering a novel Egr-1/Bim pathway by which activity deprivation induces neuronal apoptosis.

Inhibitory phosphorylation of GSK-3 by CaMKII couples depolarization to neuronal survival.

Glycogen synthase kinase-3 (GSK-3) plays a critical role in neuronal apoptosis. The two mammalian isoforms of the kinase, GSK-3α and GSK-3ß, are inhibited by phosphorylation at Ser-21 and Ser-9, respectively. Depolarization, which is vital for neuronal survival, causes both an increase in Ser-21/9 phosphorylation and an inhibition of GSK-3α/ß. However, the role of GSK-3 phosphorylation in depolarization-dependent neuron survival and the signaling pathway contributing to GSK-3 phosphorylation during depolarization remain largely unknown. Using several approaches, we showed that both isoforms of GSK-3 are important for mediating neuronal apoptosis. Nonphosphorylatable GSK-3α/ß mutants (S21A/S9A) promoted apoptosis, whereas a peptide encompassing Ser-9 of GSK-3ß protected neurons in a phosphorylation-dependent manner; these results indicate a critical role for Ser-21/9 phosphorylation on depolarization-dependent neuron survival. We found that Ser-21/9 phosphorylation of GSK-3 was mediated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) but not by Akt/PKB, PKA, or p90(RSK). CaMKII associated with and phosphorylated GSK-3α/ß. Furthermore, the pro-survival effect of CaMKII was mediated by GSK-3 phosphorylation and inactivation. These findings identify a novel Ca(2+)/calmodulin/CaMKII/GSK-3 pathway that couples depolarization to neuronal survival.