Cdk5) was reduced in cholinergic neurons of midbrain according to the single-nucleus RNA sequencing analysis. Serum levels of CDK5 also decreased in patients with Parkinson’s disease accompanied by motor symptoms. Moreover, Cdk5 deficiency in cholinergic neurons triggered paw tremors, abnormal motor coordination, and motor balance deficits in mice. These symptoms occurred along with cholinergic neuron hyperexcitability and increases in the current density of large-conductance Ca2+-activated K+ channels (BK channels). Pharmacological inhibition of BK channels restrained the excessive intrinsic excitability of striatal cholinergic neurons in Cdk5-deficient mice. Furthermore, CDK5 interacted with BK channels and negatively regulated BK channel activity via phosphorylation of threonine-908. Restoration of CDK5 expression in striatal cholinergic neurons reduced dyskinesia-like behaviors in ChAT-Cre;f/fCdk5 mice. Together, these findings indicate that CDK5-induced phosphorylation of BK channels involves in cholinergic-neuron-mediated motor function, providing a potential new therapeutic target for treating dyskinesia-like behaviors arising from neurological diseases.Disturbance of the cholinergic system plays a crucial role in the pathological progression of neurological diseases that cause dyskinesia-like behaviors. However, the molecular mechanisms underlying this disturbance remain elusive. Here, we showed that cyclin-dependent kinase 5 ( Dyskinesia-like behaviors are common neurological disorders occurring in neurological diseases such as Parkinson’s disease (PD), ataxia, dystonia, and Huntington’s disease ,2. AbnorIncreased cholinergic activity and acetylcholine levels are characteristics of PD and influence the functional output of striatal circuits ,9. Howev+ channels activity alters the excitability of striatal cholinergic neurons, which is associated with pathological process of dyskinesia and PD. For example, the decreased Kv1.3 currents induce hyperexcitability of cholinergic neurons in the striatum of PD mice [+ channels contribute to the spontaneous firing patterns of striatal cholinergic neurons [Striatal cholinergic neurons have extensive axonal fields that project locally to affect the output of striatal projection neurons , which d PD mice . Althoug neurons ,15,17, sCdk5 in the dorsal striatum disrupted the excitatory/inhibitory synaptic balance [Cdk5-conditional knockout (cKO) mice exhibited elevated intrinsic neuronal activity [Cyclin-dependent kinase 5 (CDK5) is a serine/threonine protein kinase involved in a variety of critical functions related to neural signaling pathways and neuronal activity ,19. For activity . Therefo2+-activated K+ channels (BK channels). Loss of cholinergic CDK5 induces hyperactivity of striatal cholinergic neurons and abnormal motor function in mice. Combining gene technology with electrophysiology, we demonstrate that CDK5 binds to BK channels and limits BK-mediated currents by phosphorylating threonine-908 (T908). More importantly, restoration of CDK5 expression in striatal cholinergic neurons alleviated the motor dysfunction induced by cholinergic-neuron-specific Cdk5 deficiency. Together, our pharmacological and genetic findings suggest that CDK5 signaling may represent a viable target to ameliorate dyskinesia-like disorders.Here, we report the potential roles of CDK5 in regulating striatal cholinergic neuron activity via CaCDK5 was identified as the most substantially downregulated DEG in patients with PD  and choline acetyltransferase (CHAT)  Fig. D throughAT) Fig. D and E iAT) Fig. .f/fCdk5 mice, in which exon 1 to exon 5 of Cdk5 were flanked by loxP sites, with ChAT-Cre mice to generate cholinergic-neuron-specific Cdk5-cKO mice -positive cholinergic neurons Fig. A, which ice Fig. B and C. ice Fig. D. In adddex Fig. A to C.Cdk5 loss affected motor behavior in ChAT-Cre;f/fCdk5 mice. Video data , or membrane capacitance (Cm) between the 2 groups of mice  were unaltered , delayed rectifier K+ currents (kI currents), and calcium-activated K+ currents (BK currents) [kI currents  . We obsents Fig. B or IA cnts Fig. C. HoweveBTX Fig. E; thus, channels . We therice Fig. B and C. Cdk5 deficiency contributes to the elevation of the BK-channel-mediated current density, we examined the association between CDK5 and BK channels. Coimmunoprecipitation and Western blotting data suggested that CDK5 binds directly to the BK channel in striatal cholinergic neurons -eGFP (BK-eGFP) and Cdk5-Flag plasmids and transiently transfected them into Neuro 2A (N2A) cells , cotransfected them with or without the Cdk5-Flag plasmid into N2A cells, and performed electrophysiology recordings. As expected, the increase in the BK current density induced by BK-eGFP transfection was blocked by cotransfection of BK-eGFP and Cdk5-Flag in N2A cells. More interestingly, there was no difference in the BK current density among N2A cells transfected with BK-eGFP and Flag, T908ABK-eGFP and Flag, or T908ABK-eGFP and Cdk5-Flag  into the striata of ChAT-Cre;f/fCdk5 mice and an AAV-expressing mCherry into the striata of ChAT-Cre mice , beta (13 to 35 Hz), low-gamma (36 to 65 Hz), and high-gamma (66 to 95 Hz) oscillations were decreased in the striata of ChAT-Cre;f/fCdk5 mice; these decreases were significantly reversed by Cdk5 overexpression  were crossed with f/fCdk5 mice  [f/fChAT-Cre;Cdk5 mice. BAC-eGFPChAT mice  were crossed with f/fCdk5 mice and f/fChAT-Cre;Cdk5 mice respectively for the electrophysiology recording of cholinergic neurons.oratory)  to generPatients with PD with motor disorders were enrolled in this study. Serum samples were collected from age- and race-matched controls and patients. All patients and controls were 50 to 80 years old and Asian race. The average age was 67.2 years old for patients with PD and 67.2 years old for HCs. The study with patients is approved by the Ethics Committee of Affiliated Brain Hospital of Nanjing Medical University with number 2021-KY007-01.CHAT and ACHE) and processing snRNA-seq data analysis as reported [Major cell types from the midbrain of patients with PD and HCs were identified and annotated by interrogating the expression patterns of known marker genes: cholinergic neurons  vector was constructed using Cdk5 with a 3×Flag. The pAAV-EF1α-DIO-mCherry  was used as control. The pAAV-EF1α-DIO-Cdk5-mCherry was bilaterally injected into the striatum of f/fChAT-Cre;Cdk5 mice. The pAAV-EF1α-DIO-mCherry was bilaterally injected into the striatum of ChAT-Cre and f/fChAT-Cre;Cdk5 mice. Both of AAV vectors were packaged into AAV2/9 serotype by OBiO Technology .To overexpress 12 viral particles/ml) or the rAAV-ChAT-EGFP (as control)  was bilaterally injected into the striatum of f/fCdk5 mice. These AAV vectors were packaged into AAV2/9 serotype by BrainVTA .To eliminate the striatal cholinergic neuron CDK5, the rAAV-ChAT-Cre-EGFP  . All mouFootprint test was done as previously described . Mice weGrip strength test was conducted using the grip test system . Mice weMice at the age of P21 to P30 were anesthetized. As described previously , the braCell-attached recordings were applied to measure the spontaneous firing APs of cholinergic neurons as described before .Rin), membrane time constant (Tau), and membrane capacitance (Cm) were recorded.Whole-cell recordings were performed from striatum cholinergic neurons with a MultiClamp 700B amplifier as described previously . Glass m+ currents and K+ currents were recorded in voltage-clamp mode. For Na+ currents recording, the bath solution contained tetraethylammonium chloride  and CsCl , the Na+ in intracellular solution was replaced by Cs+. The total K+ currents were recorded in the presence of tetrodotoxin . kI currents were recorded at voltages of −120 and +10 mV for 200 ms, followed by −80- to +70-mV voltages, with 10-mV increments, and 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid  was added to electrode internal fluid to inhibit calcium-dependent potassium currents [AI currents were inactivated at voltages of −120 and +10 mV for 200 ms and then calculated by subtracting the remaining K+ currents from total K+ currents.Nacurrents . IA currBK channel activity was recorded under voltage-clamp mode as previously reported . Before Calcium currents were recorded according to previously used protocol .Data were analyzed using MATLAB , Clampfit 10 (Molecular Devices), and MiniAnalysis software (Synaptosoft).The immunohistochemistry staining was performed as previously . Briefly2. After cultured for 24 h, the N2A cells were transfected with plasmids using Lipofectamine 3000  for 12 h and replaced with fresh medium for another 48-h culture.Cell culture and transfection were done as before . N2A celCdk5 was provided by X. Guo . Full-length mslo (encoding BK) (NM_001253378.1) cDNA was amplified from the cDNA of primary neuronal cells using the following primer set: sense, 5′- AAACTCGAGATGGCAAACGGTGGCGGCGGCGGCGGCGGC-3′; antisense, 5′- AAAGCGGCCGCCACATTCATCTTCAACTTCTCTGAT-3′. In these primers, Xho I and Not I restriction site sequences have been underlined. The polymerase chain reaction products were cloned into the pEGFP-N1 vector. BK carrying the S716A mutation was amplified using the following primer set: sense, 5′-GGGCATGAGGAACGCGCCCAACACCTC-3′; antisense, 5′-GAGGTGTTGGGCGCGTTCCTCATGCCC-3′. BK carrying the S720A mutation was amplified using the following primer set: sense, 5′-CTCGCCCAACACCGCCCCGAAGCAGAT-3′; antisense, 5′-ATCAGCTTCGGGGCGGTGTTGGGCGAG-3′. BK carrying the T908A mutation was amplified using the following primer set: sense, 5′-CTAATTCCCAAGGATTCGCACCTCCTGGAATGGAC-3′; antisense, 5′-GTCCATTCCAGGCGGTGCGAATCCTTGGGAATTAG-3′. BK carrying the T1112A mutation was amplified using the following primer set: sense, 5′-CCCACCTCAGCGCCCCCAGCCAG-3′; antisense, 5′-CTGGCTGGGGGCGTGAGGTGGG-3′. All constructions were ensured by sequencing.The pcDNA3.1-3X-Flag-Immunoprecipitation (IP) and Western blot (WB) were conducted as previously described . BrieflyThe EMG of leg muscles were recorded as described before . The gasMice were deeply anesthetized with 1.5% to 2% isoflurane gas/oxygen mixture and fixed on the stereotactic apparatus ,68. ThenFor LFP recordings, signals were amplified , filtered at 0.1 to 250 Hz, and sampled at 1 kHz. The analysis of the LFP signals was conducted by a program written in MATLAB.Spectral power was obtained by using MATLAB’s wavelet method (MathWorks), continuous wavelet transform, and Morlet wavelets . Power st test was performed for 2 independent group comparisons. One-way analysis of analysis of variance (ANOVA) followed by Tukey’s post hoc test was used for multiple normally distributed data. Two-way ANOVA was used for dataset with 2 factors, followed by Bonferroni’s multiple comparison test. *P < 0.05 was considered to be statistically significant.Data were analyzed by experimenters blinded to the mice genotype. GraphPad Prism 8 was applied for statistical analysis. All data were displayed as means ± SEM. Unpaired 2-tailed Student’s


Persistent left superior vena cava (PLSVC) is a congenital venous anomaly. It is frequently associated with other cardiac anomalies. The presence of dual superior vena cava is due to the lack of development of the left cardinal vein in utero. The coronary sinus gets dilated as a result of increased blood flow to the right heart and may be seen on echocardiography. This case describes a 50-year-old lady who presented to the emergency department with lightheadedness, nausea, and vomiting for one day, and her electrocardiogram showed a heart rate of 30 beats per minute. A temporary pacemaker was placed. She had a history of asymptomatic PLSVC diagnosed six months ago through percutaneous coronary intervention. A permanent pacemaker was passed through PLSVC to access the right ventricle and she was discharged home after five days of an uneventful hospital course. Clinicians should be aware of this rare congenital anomaly and its potential complications, particularly in patients with unexplained syncope or bradycardia. Further research is needed to better comprehend the clinical presentation, diagnostic evaluation, and management of PLSVC-related cardiac abnormalities. Persistent left superior vena cava (PLSVC) occurs because of the congenital anomalous presence of superior vena cava (SVC) on the left. Although the exact mechanism of PLSVC is unknown, it is hypothesized that the lack of development of the left cardinal vein in utero is responsible for the dual SVC . PLSVC cA 50-year-old, stay-at-home lady, presented to the emergency department of a tertiary care hospital with light-headedness, nausea, and vomiting for one day. She was a known case of diabetes and hypertension for 10 years. She underwent a percutaneous coronary intervention (PCI) which showed blockage in the obtuse marginal (OM) branch of the left circumflex artery. The rest of the coronary vessels were normal. A stent was placed in the OM vessel six months ago. During the procedure, incidental asymptomatic PLSVC was found, and observation was advised.On examination, she appeared sick but was oriented to time, place, and person. She was afebrile with a blood pressure of 130/80 mmHg, pulse rate of 40 beats per minute, and respiratory rate of 22 breaths per minute. An electrocardiogram showed sinus bradycardia  insertion, cardiac resynchronization therapy, or pacemaker implantation, electrode fixation becomes challenging due to the tortuous course of PLSVC. CVC insertion without fluoroscopic guidance can cause angina, hypotension, and heart perforation. In the case of constricted CS, catheterization might lead to significant complications, such as arrhythmias, cardiogenic shock, and cardiac tamponade. Because of the steal effect of the hemiazygos venous system related to PLSVC, retrograde cardioplegia may fail despite PLSVC clamping . The knoTo conclude, PLSVC can present variable consequences. An enlarged CS on echocardiography requires confirmation with saline contrast for possible PLSVC. A high index of suspicion is needed whenever the guide wire inserted via the left subclavian vein takes a left-sided course. Besides technical difficulties associated with right heart access, additional risks related to PLSVC should be considered.


Neisseria meningitidis serogroup W (NmW) galactose–sialic acid  heteropolysaccharide binds lead ions effectively, thereby demonstrating the potential for its use in environmental decontamination applications. In this study, computational analysis of the NmW capsule polymerase galactosyltransferase (GT) domain was used to gain insight into how the enzyme could be modified to enable the synthesis of N-acetylgalactosamine–sialic acid  heteropolysaccharide. Various computational approaches, including molecular modeling with I-TASSER and molecular dynamics (MD) simulations with NAMD, were utilized to identify key amino acid residues in the substrate binding pocket of the GT domain that may be key to conferring UDP-GalNAc specificity. Through these combined strategies and using BshA, a UDP-GlcNAc transferase, as a structural template, several NmW active site residues were identified as mutational targets to accommodate the proposed N-acetyl group in UDP-GalNAc. Thus, a rational approach for potentially conferring new properties to bacterial capsular polysaccharides is demonstrated.Heavy metal contamination of drinking water is a public health concern that requires the development of more efficient bioremediation techniques. Absorption technologies, including biosorption, provide opportunities for improvements to increase the diversity of target metal ions and overall binding capacity. Microorganisms are a key component in wastewater treatment plants, and they naturally bind metal ions through surface macromolecules but with limited capacity. The long-term goal of this work is to engineer capsule polymerases to synthesize molecules with novel functionalities. In previously published work, we showed that the  Biosorpngitidis , and ourngitidis ,9.In previous work, the lead-binding affinity of NmW capsular polysaccharides was demonstrated . SolutioW gene, is a 120 kDa glycosyltransferase comprised of an N-terminal GT domain, an intervening sequence, and a C-terminal sialyltransferase domain [The NmW capsule polymerase, encoded by the SiaDe domain –15. The e domain ,16. Meche domain .Bacillus subtilis, Bacillus anthracis, Staphylococcus epidermis, and Staphylococcus aureus [The structures of several members of the BshA family, also classified as GT4 family enzymes, are available. These include family members from pathogenic and non-pathogenic species such as s aureus –20. The s aureus . Still, The studies described here report on sequence analysis, structure prediction, and molecular dynamics of the NmW-GT. Our results have identified several putative ligand binding residues that confer substrate specificity. Simulations and molecular docking could guide the engineering of glycosyltransferases for the synthesis of novel materials.2.2.1.N. meningitidis serogroup W capsule polymerase (NmW) (SiaDW) was obtained from NCBI [Neisseria meningitidis [Bacteroides ovatus [The genomic sequence of rom NCBI   [The galactosyltransferase domain of the NmW capsule polymerase was modeled with I-TASSER –33. The e: 6D9T) . Its RMSe: 6D9T) –36 was ue: 6D9T) ,38. To fe: 6D9T) . The NmWe: 6D9T) ,41.2.3.2 and probe radius of 1.4 Å.Docking of UDP-hexoses and the model was conducted with DOCK6 –45 as foSpheres were created with sphgen and clusters falling within the proximity of the active site were selected using sphere selector. The default parameters for sphere generation with sphgen were used as follows: spheres were generated to cover the outside surface (R), all points were used (X), the generation of large spheres was suppressed, and maximum and minimum sizes of the spheres were set at 4.0 Å and 1.4 Å, respectively. The location of the active site was estimated firstly by structural alignment of the NmW-GT model with the BshA structure identified by the I-TASSER server in UCSF-Chimera, and a region within 10 Å from the ligand (UDP) was selected with the Zone tool, saved in mol2 format, and used as an input for the sphere selector program. The input for the grid program was generated with Showbox; all spheres in the cluster encompassing the active site were included and the spacing was set at 8 Å.The Grid program was then used to generate grids using the AMBER_parm99.defn parameters. The docking grid was generated with a spacing of 0.4 Å, and energy scoring was used with the exponent of attractive and repulsive Lennard–Jones terms for VDW set at the default values of 6 and 12, respectively. The coefficient of the dielectric was also set at the default value of 4.0.Finally, the Dock6.9 program was used to conduct flexible docking on the Stampede2 supercomputer at Texas Advanced Computing Center (TACC), The University of Texas at Austin. Flexible docking searching method was utilized with essentially the default parameters as follows: the minimum number of atoms in an anchor was set to 5; pruning of conformers was used with a maximum of 1000 orientations, 100 clusterheads were retained, and a cutoff of 1.0 was used as the pruning conformer score scaling factor; internal energy scoring was used and the VDW exponent was set to the default value of 12 kcal/mol and the cutoff for pruning conformers was set at 100.0 kcal/mol; minimization of the ligand, anchor, and flexible growth during docking was conducted with the default parameters of 500 iterations, maximum cycles of 1, score convergence of 0.1, cycle convergence of 1.0, translation step size of 1.0, rotation step of 0.1, and torsional step of 10.0.2.4.The systems for molecular dynamics simulations were prepared with CHARMM-GUI using the default parameters –51. To sThe simulation steps  were performed sequentially with NAMD ,55. The 2.5.UCSF-Chimera , VMD , was the top structure with the highest C-score of 0.31 (a measure of confidence in the model) and shared 17% sequence identity with the NmW-GT domain. This protein plays a functional role in bacillithiol biosynthesis. Both enzymes are members of the GT4 CAZy family  . It has S. aureus BshA) that is identified as forming hydrogen bonds with GlcNAc (Ndg402) in S. aureus BshA is highly conserved in BshA enzymes. Two residues that interact directly with GlcNAc in this motif (E and F) are also conserved in NmW-GT , and they map to the same regions identified through experimental data by other authors. The region containing the ESFG motif  as the template. The structure of S. aureus BshA was solved in the presence of UDP and in the presence of UDP and N-acetylglucosamine (GlcNAc) [The NmW-GT domain model used in these studies was generated with I-TASSER –33 using(GlcNAc) . The str(GlcNAc) . Among tEquilibration of the system was confirmed via RMSD analysis within UCSF-Chimera . FurtherA dynamic cross-correlation matrix (DCCM) was constructed to determine the regions that move in concert during the trajectory . Inciden4.Neisseria meningiditis serogroup W capsule polymerase was modeled to inform experimental studies that seek to alter the substrate specificity of this domain. We identified putative active site residues through structure prediction approaches that incorporate de novo and homology modeling and sequence alignments. Molecular dynamics simulations also revealed regions of fluctuation that surround the active site region and that might influence enzyme conformations and hence activity.Heavy metal environmental pollution remains a challenging problem that could potentially be alleviated through biosorption. Microbial polysaccharides have been shown to effectively bind lead in water. To enhance chelation and hence the capacity for bioremediation, biosynthetic routes for capsular polysaccharides with desired properties are currently under development. The GT domain of the In the context of the full-length NmW capsule polymerase, others have shown that P310G mutation leads to a switch in nucleotide donor specificity from UDP-Gal to UDP-Glu . This ca7E motif of the GT4 family mediates catalysis. Still, while E280 is proximal to the reactive bond, E288 is positioned closer to the uracil group, which is consistent with the observation that the mutation of the initial E in the AceA α-mannosyltransferase from Acetobacter xylinum, also a GT4 enzyme, leads to a complete loss of enzymatic activity while mutants of the second E retain some residual activity [2, double displacement, and substrate-assisted SNi. Recent crystallography and enzymatic studies have definitively ruled out two of the three. H118 was shown to play a critical role in stabilizing the oxocarbenium-like intermediate that develops by the SNi-like mechanism. Although the NmW-GT domain has been characterized as a retaining glycosyltransferase, the mechanistic details of how this transfer takes place have not been determined due to the lack of a three-dimensional structure. Given that H118 and the aforementioned residues except T120 are conserved in NmW-GT, the present work alludes to a similar catalytic mechanism for this domain.Mutation of either H118, T120, K209, E280, or E288 led to the complete loss of BshA catalytic activity, suggesting a role for these residues in the reaction mechanism . This isactivity . This moactivity . MD simuactivity . In thisSome BshA mutations have been reported to eliminate enzymatic activity despite not being proximal to the substrate reacting centers . The resSupplementary Material


A molecular understanding of heterochrony was first established by genetic mutant analysis in the nematode C. elegans, revealing a genetic pathway that controls the proper timing of cellular patterning events executed during distinct postembryonic juvenile and adult stages2. This genetic pathway is composed of a complex temporal cascade of multiple regulatory factors, including the first-ever discovered miRNA, lin-4, and its target gene, lin-14, which encodes a nuclear, DNA-binding protein4. While all core members of the pathway have homologs based on primary sequences in other organisms, homologs for LIN-14 have never been identified by sequence homology. We report that the AlphaFold-predicted structure of the LIN-14 DNA binding domain is homologous to the BEN domain, found in a family of DNA binding proteins previously thought to have no nematode homologs5. We confirmed this prediction through targeted mutations of predicted DNA-contacting residues, which disrupt in vitro DNA binding and in vivo function. Our findings shed new light on potential mechanisms of LIN-14 function and suggest that BEN domain-containing proteins may have a conserved role in developmental timing.Heterochrony is a foundational concept in animal development and evolution, first introduced by Ernst Haeckel in 1875 and later popularized by Stephen J. GouldC. elegans LIN-14. BLAST sequence analysis readily recovered LIN-14 sequence homologs within the nematode phylum, but not outside. Sequence alignment of all 139 LIN-14 homologs defined a conserved 149 amino acid residues long region within a minimal LIN-14 protein fragment previously shown to bind DNA and to be sufficient to rescue lin-14 mutant phenotypes   5,8.C. elegans, the BEN domain is found in proteins across the animal kingdom, many of which are involved in transcriptional regulation and chromatin organization8. Since BEN domain proteins have been crystallized together with DNA target sequences5, we were able to predict that several basic, positively charged residues in the fifth (and last) α helix of the LIN-14 BEN domain contact DNA directly  in lin-14 mutant animals9. One such candidate is the neuropeptide-encoding gene nlp-45.While BEN domains have not previously been described in directly . To expenlp-45 locus  Arg mutant animals are indistinguishable from lin-14(ma135) null mutant animals; they are sterile, have a dumpy appearance, a protruding vulva, display precocious alae, and show de-repression of nlp-45 gene expression at the improper time, during the first larval stage (Next, we used CRISPR/Cas9 genome engineering to introduce the four arginine mutations into the endogenous al stage -F.C. elegans protein structures from AlphaFold revealed several other C. elegans proteins with predicted similarities to the BEN domain (C. elegans10. Another previously uncharacterized protein with two putative BEN domains, F12F6.1 (9.Structural homology searches using DALI against all predicted N domain . The onl F12F6.1 , also sh5, it is conceivable that, in addition to transcriptional regulation, LIN-14 may also play a role in chromatin organization. Since several non-nematode BEN domain-containing proteins have, like nematode LIN-14 and SEL-7, roles in temporal patterning8, such a function may have been the ancestral role of BEN domain proteins.The structural deorphanization of LIN-14 as a BEN domain-containing protein provides new vistas on both LIN-14 protein function as well as BEN domain proteins in general. Since many BEN domain proteins are involved in controlling chromatin architectureMMC1


This study aimed to validate a specialized competency framework for industry pharmacists and assess correlates related to the competency domains in a pilot sample.A team of experts assessed the old framework and improved its content validity after a thorough literature review, using the Delphi technique. Domains and their respective competencies and behaviors were re-defined in the framework. Afterward, a web-based cross-sectional study was carried out between March and October 2022, enrolling a convenient sample of ten industry pharmacists who worked in Lebanese pharmaceutical plants. Participants were contacted through the Syndicate of the Pharmaceutical Industries in Lebanon.The specialized competency framework for Lebanese industry pharmacists comprised seven domains. Behavioral items had appropriate loading on their respective factors, which could involve one, two or three competencies. Cronbach alpha values for all domains were close to one, showing appropriate reliability. Each domain was correlated with at least another one, except for domains related to pharmaceutical and industrial development and emergency preparedness, which were not correlated with other domains. The lowest confidence was found in the research and development domain, particularly among participants with only a PharmD.This study validated the specialized competency framework for Lebanese industry pharmacists. Some domains, specifically those related to industrial development and emergency preparedness, were found to diverge from others. Therefore, it would be recommended to include additional education in the emergency preparedness, research and development fields and to integrate industry-specific skills, courses, and training programs into academic curricula. Furthermore, specialized postgraduate degrees may be necessary to produce practice-ready pharmacists to operate effectively in this vital setting.The online version contains supplementary material available at 10.1186/s40545-023-00602-8. Professional competence definition has long been debated. However, a stable concept is that a set of skills, including knowledge, abilities, and behaviors, indicates a specific competency that leads to better performance in a particular career. Competency-based education is currently trending as it allows universities to produce practice-ready professionals. The competency-based model is thus a widely used approach for defining professional effectiveness at individual and organizational levels .Pharmacy competencies often comprise pharmaceutical knowledge, leadership/managerial aspects, and professional/functional skills . DevelopWorldwide, several countries are working to strengthen the competencies of their pharmacists to serve some specific employment tasks –9. The dIn high-income nations, competency-based developmental frameworks are becoming more popular in industrial careers and are widely used to create requirements for training, education, and career advancement , 13, 14.In developing countries, the landscape of pharmacy practice often differs from that of developed nations. While some developing countries have made efforts to develop core competency frameworks for pharmacists, there has been a limited focus on developing specialized frameworks specifically tailored for professionals working in pharmaceutical manufacturing , 17. In In Lebanon, the mismatch between educational curricula and the job market needs is unequivocal in all professional sectors, including industrial pharmacy. A specialized competency framework adapted to the local context was previously suggested by the Order of Pharmacists of Lebanon  . It compThis framework has not been validated or piloted, although it would help bridge the gap between traditional pharmacy education and the constantly evolving needs of the pharmaceutical industry . It woulIn light of the above, it was deemed necessary to validate and pilot the competency framework for industry pharmacists after adding a section related to emergency preparedness to complement the set of competencies required from industry pharmacists, as suggested by the FIP revised framework . TherefoInspired by a French model , the iniDomain 0: Research and Development ; Domain 1: Pharmaceutical and Industrial Development ; Domain 2: Analytical Development ; Domain 3: Industrial Pharmaceutical Production ; Domain 4: Quality Management .Industry stakeholders confirmed the suggested document through semi-structured interviews. These stakeholders also expressed the lack of relevant postgraduate studies locally, leading graduates to pursue education abroad or undergo onsite training for several months.A team of experts that comprised four academics and two industry pharmacists gathered and reviewed the content of the previously suggested industrial pharmacy framework. After a thorough literature review, domains, competencies, and behaviors (items) were reviewed, updated, restructured, and adapted to the Lebanese setting. Two competencies of the previously suggested tool, i.e., Quality Assurance and Quality Control, which were under the same domain , were extracted and put under a new domain termed “Quality Assurance and Control” (sixth domain). In the absence of research and development for innovative drugs in the country, this framework did not include a section on the different phases of clinical trials, as industry pharmacists in Lebanon are not involved in this role, since R&D departments exclusively focus on the development of generic medications.Furthermore, since the pandemic occurred and multiple crises hit the country after the previous framework was diffused, a seventh domain (Domain 6: Pharmacist Preparedness and Response in Emergency Situations) inspired by several studies and frameworks was added and included competencies related to the pharmacist during emergencies –30.Using a Delphi technique, the framework was circulated to experts for more than five rounds until a consensus of 90% or more was reached on all items. The Delphi technique is a structured communication method used to gather and analyze the opinions of a group of experts on a particular topic . Its goaDomain 0: Research and Development ;Domain 1: Pharmaceutical and Industrial Development ;Domain 2: Analytical Development ;Domain 3: Industrial Pharmaceutical Production ;Domain 4: Quality Assurance and Control ;Domain 5: Quality Management ;Domain 6: Pharmacist Preparedness and Response in Emergency Situations .The finalized framework was subsequently adapted to be administered to industry pharmacists through standardized online questionnaires.To validate the structure of the framework, a web-based cross-sectional study from March to October 2022 enrolled a convenient sample of ten industry pharmacists working in Lebanese pharmaceutical plants. Participants were contacted through the Syndicate of the Pharmaceutical Industries in Lebanon (SPIL). Respondents were briefed about the topic and the different aspects of the questionnaire in the introductory section of the questionnaire. They gave their consent before proceeding to the survey. All industry pharmacists with managerial positions and living in Lebanon were eligible to participate. Anonymity and confidentiality were ensured across the entire data collection process.The questionnaire was in English, a language commonly spoken by healthcare professionals in Lebanon. It comprised three sections. In the first section, participants were asked about their general sociodemographic data, including their age, gender, nationality, area of work, university of graduation, highest educational level, years of experience, number of working hours per day, and number of working days per week. The second part consisted of the scale-based framework, including the seven domains divided into competencies and related behaviors. The last section involved four questions about, where these competencies were acquired  coefficient, Bartlett’s sphericity test, and the total percentage of variance explained were reported for each analysis. In addition, Cronbach alpha values were calculated for each competency to assess internal consistency (reliability); Cronbach alpha assesses reliability by comparing the amount of shared variance, or covariance, among the items that make up an instrument to the amount of total variance . Acceptap value less than 0.05 was considered significant.Afterward, a descriptive analysis was performed using the counts and percentages for categorical variables and means and standard deviations for continuous measures. Means were compared using the Mann–Whitney test, while percentages were compared using the Chi-square test (or the Fisher exact test when necessary) to assess correlates of competencies. A Table Table Domain 0: Research and Development ;Domain 1: Pharmaceutical and Industrial Development ;Domain 2: Analytical Development ;Domain 3: Industrial Pharmaceutical Production ;Domain 4: Quality Assurance and Control ;Domain 5: Quality Management ;Domain 6: Pharmacist Preparedness and Response in Emergency Situations .Moreover, Cronbach alpha values for all domains were close to one, showing appropriate reliability Table .Table The descriptive results in Table In bivariate analysis, pharmacists with only a PharmD reported the lowest confidence in Domains 0, 1, and 2 , while those who graduated from University 2 reported low confidence in Domain 5  , while continuing education programs had a lower correlation ; however, gaining competencies by experience was associated with lower confidence  (results not shown).Finally, the median percentages for the declared sources of competencies were 20% for undergraduate studies, 55% for postgraduate studies, 17.5% for continuing education, and 87.5% for experience. The highest confidence in overall competencies was found to be best correlated with postgraduate studies . This result may be explained by the lack of training activities relevant to this domain in internship programs, which, in turn, is due to the scarcity of research and development activities in many Lebanese pharmaceutical plants . Moreovep < 0.05). Knowing that PharmD is the gate to clinical practice, pharmacists earning this degree may not be eligible to carry out industrial processes, research and pharmaceutical analysis procedures. Consequently, their role may be limited to the follow-up of routine production processes and quality issues. In educational institutions, additional efforts are needed to recognize the importance of industrial pharmacy. Over the last two decades, the role of the industry pharmacist in developed countries has expanded to cover multiple domains and was recognized as a vital professional role in the provision of healthcare [In our study, pharmacists with only a PharmD degree had the lowest declared confidence in domains related to research and development and industrial and analytical development  and create postgraduate programs specific to emergency preparedness. However, the efficacy of these measures relies on their integration into a national strategy that encompasses all stakeholders, including pharmacists from different sectors. Collaborative efforts between regulatory bodies, industry associations, academic institutions, and government agencies are crucial for a coordinated approach. This approach allows for the development of standardized guidelines, regulations, and protocols to strengthen emergency preparedness across the pharmaceutical industry. Furthermore, engaging with pharmacists from diverse sectors facilitates the sharing of experiences and best practices, ensuring comprehensive emergency response capabilities that address the unique challenges faced by each pharmacy setting. By adopting this collective approach, the emergency preparedness of industry pharmacists can be improved, ensuring uninterrupted medication supply and bolstering the overall resilience of the pharmaceutical industry during crises.Pharmacy schools principally direct students to the clinical aspects of the pharmacy profession, which may affect their willingness to follow an industrial career path . This faThe median percentage for the declared source of competency was the highest for experience (87.5%); however, competencies acquired through experience were correlated with lower confidence in performing tasks. The highest confidence in overall competencies was found to be best correlated with postgraduate studies. Furthermore, continuing education was the lowest declared source of competency (17.5%) and had a weak correlation with confidence. These findings indicate that postgraduate programs have contributed to preparing competent industry pharmacists able to perform various industrial tasks appropriately. Nevertheless, well-structured internship programs are essential to prepare industry pharmacists with high-level competencies in all domains. Although such programs are implemented in some Lebanese pharmacy schools, a limited number of students can be enrolled, likely due to the lack of communication between universities and pharmaceutical plants and the limited number of local pharmaceutical plants . SimilarIn some countries, a tangible academic collaboration exists between several US pharmacy schools and pharmaceutical manufacturing plants aiming to provide students with pharmacy fellowships and prepare them for the pharmaceutical industry profession . AccordiDespite its importance, this pilot study has several limitations. A selection bias is possible, given the low number of participants in the assessment; nevertheless, the adequate structure, good reliability, and significant associations and differences found are not expected to change with larger samples. Moreover, an information bias is also possible, given the length of the questionnaire and possible fatigue of participants added to the self-declared nature of the information. However, this bias would be non-differential and only direct the results toward the null. Finally, no multivariable analysis could be conducted to decrease confounding during the assessment of associations. Thus, further large-scale studies are recommended to overcome these limitations.This study validated the specialized competency framework for Lebanese industry pharmacists. Some domains, specifically those related to industrial development and emergency preparedness, were found to diverge from others. Therefore, it would be recommended to include additional education in the emergency preparedness, research and development fields and to integrate industry-specific skills, courses, and training programs into academic curricula. Furthermore, specialized postgraduate degrees may be necessary to produce practice-ready pharmacists to operate effectively in this vital setting.Additional file 1. Advanced Competencies for Industry Pharmacists questionnaire.


The topology type and the functionalization of pores play an important role in regulating the performance of covalent organic frameworks. Herein, we designed and synthesized the covalent organic framework with hetero-environmental pores using predesigned asymmetrical dialdehyde monomer. According to the results of structural characterization, crystallinity investigation, and theoretical calculation, the hetero-environmental pores of the obtained framework are regarded as the alternant arrangement. The distinctive hetero pore structure leads the designed material to show more advantages as compared with control materials in loading both hydrophobic and hydrophilic antibiotics for wound healing. This dual-antibiotic strategy can expand the antibacterial range as compared with the single antibiotic one, and reduce the generation of drug resistance. In summary, this strategy for designing covalent organic frameworks with hetero-environmental pores can extend the structural variety and provide a pathway for improving the practical application performance of these materials. The characteristics of the pores are vital for controlling the performance of covalent organic frameworks, but obtaining different chemical environments in different pores is challenging. Here, the authors report the development of covalent organic frameworks with differing pore environments. COFs can be constructed by integrating components into two-dimensional (2D) polymers to form ordered nanopores, which are designable, adjustable, and modifiable through pore engineering5. Meanwhile, COFs possess precise spatial structures and regular block distributions, which result in forming periodic columnar π-arrays and directional one-dimensional (1D) channels7. The structural research on COFs mainly focuses on the topology of the framework and the surface engineering of the channels9. The different topological structures of the frameworks generate various shapes and sizes of the pores, which can be obtained by elaborately designing the building blocks with different sizes and specific symmetries15. The channel surface engineering of COFs is mainly concentrated on regulating the chemical composition of the framework and the functionalization of the pores through the process of pre-synthesis modification or post-synthesis functionalization19. Since Zhao and co-workers reported a dual-porous COF with the periodic and orderly distribution of micropores and mesopores in 2014, a series of COFs with multiple pores in one framework have been prepared20. These reported heteroporous COFs exhibit some specific interesting properties24. For example, Ma and co-workers proved that dual-porous COFs are more advantageous than single-porous COFs in the enzyme host-catalyzed organic reaction25. In their design, the large hexagonal pores are employed to encapsulate enzymes as a catalytic space, while the small triangular holes are used as channels for transporting reagents and products. The coexistence of pores with multiple sizes can effectively minimize diffusion barriers. The heteroporous COFs with various pore shapes and sizes have been well-studied, however, the chemical environment of the pore channels is homogeneous. The COFs with different chemical environments of pore channels are seldom investigated.Covalent organic frameworks (COFs) have become a powerful platform for the design of functional materials since the pioneering work has been reported by Yaghi and co-workers in 200526. By combining antibiotics with different/opposite properties can overcome resistance mechanisms of bacteria and increase the treatment effectiveness27. For complex infections caused by drug-resistant bacteria, the combination of antibiotics is an emerging and important strategy to effectively combat drug-resistant bacterial infections. However, the compatibility of different drugs with disparate properties (such as the different hydrophilicity) limits the combination of drugs29. In recent years, various antibacterial materials and strategies have been developed30. Shi et al. incorporated hydrophilic (–)-epigallocatechin-3-O-gallate (EGCG) in the shell of pH-responsive mixed shell polymeric micelle, and loaded hydrophobic ciprofloxacin in the core. It is proved that the co-delivery system of hydrophilic/hydrophobic drugs possesses balanced biofilm dispersal and killing31. Fan et al. redesigned the inner surface of ferritin drug carrier (ins-FDC) nanocarrier as a drug delivery platform and used the urea-thermal incubation loading method to maximize the loading capacity of hydrophilic and hydrophobic drugs at the same time. The result shows that the dual-drug loaded system exhibits a higher synergistic antitumor effect than the single-drug loaded ones and efficiently overcomes the tumor cell drug resistance32. The co-delivery system is imperative and should be further explored. Previous studies demonstrate that COFs can serve as carriers to upload and deliver drugs for therapeutic applications34. If the designed COF contains unique hetero-environmental pores, it can be predicted to be an ideal co-delivery carrier for drugs.Antibiotic-resistant bacteria pose a significant threat to human health. The development of antibiotics lags behind the evolution of bacterial resistance, highlighting the urgent need to effectively utilize existing antibiotics. Aminoglycoside/beta-lactam combinations exhibit synergistic effects and are the most commonly used combinations in clinical practiceIn this work, we designed and synthesized an imine-linked COF, DEG-HEP-COF, by connecting 5,10,15,20-tetrakis(4-aminophenyl)porphyrin (TAPP) and dialdehyde monomer with both hydrophobic alkyl chain and hydrophilic alkoxy chain in one molecule. Meanwhile, the COFs containing only hydrophilic chains (DEG-COF) or hydrophobic chains (HEP-COF) were constructed and utilized as control samples to evaluate the structures and properties of DEG-HEP-COF. Furthermore, in order to investigate the effect of the uniformity of pore distribution on the material properties, another control sample (DEG+HEP-COF) was prepared via the multi-component copolymerization method, which possesses the random and heterogeneous porous channels of hydrophoblity and hydrophility. Powder X-ray diffraction (PXRD) measurement, nitrogen adsorption–desorption experiment, and simulation calculation results indicate that DEG-HEP-COF possesses hetero-environmental pores, which are arranged in periodic arrays similar to the chess board. Based on its unique hetero-environmental pores, the DEG-HEP-COF was used to load hydrophobic levofloxacin (Lev) and hydrophilic vancomycin (Van) to explore the relationship between the pore environment and the loading/relasing behaviors of antibiotics. The experimental results show that the dual-antibiotic delivery strategy by using the DEG-HEP-COF carrier can expand the antibacterial range as compared to a single antibiotic, and delay or reduce the generation of drug resistance. Therefore, this kind of COFs containing hetero-environmental pores can be similarly prepared by designing monomers elaborately, and this synthesis strategy can expand the structural and functional variety of COF materials .1H NMR, 13C NMR, and high-resolution mass spectra (HR-MS)  (1/9/2 in volume ratio), 1,4-dioxane/ethanol/acetic acid (6 M) (1/9/2 in volume ratio), or mesitylene/ethanol/acetic acid (6 M) (1/9/2 in volume ratio) at 120 °C for 3 d. The variety of optimized reaction conditions results from the adaptability of the amphiphilic monomer to different solvents, which reduced the difficulty in synthesis condition screening. Here, the mixture of o-dichlorobenzene/ethanol/acetic acid (6 M) (1/9/2 in volume ratio) was chosen as the reaction condition of DEG-HEP-COFs that was used to investigate the subsequent structure and performance. The control COFs were obtained under the optimized preparation conditions of ethanol/acetic acid (6 M) (5/1 in volume ratio) for DEG-COF, o-dichlorobenzene/ethanol/acetic acid (6 M) (7/3/2 in volume ratio) for HEP-COF, and o-dichlorobenzene/ethanol/acetic acid (6 M) (5/5/2 in volume ratio) for DEG+HEP-COF at 120 °C for 3 d, respectively. Then, the FT-IR, solid-state 13C NMR measurement, X-ray photoelectron spectroscopy (XPS), and elemental analysis were utilized to confirm the structures of these COFs. FT-IR spectra show stretching vibration peaks at 1601 cm–1 for all these COFs, which can be assigned to the C=N bond and demonstrates the formation of imine linkages , suggesting the high imine condensation degree. The phenomenon in DEG+HEP-COF is owing to the reactivity difference between DEG-CHO and HEP-CHO, which may cause incomplete reaction of the terminal aldehyde groups. Meanwhile, several peaks in the range of 2850–3000 cm–1 can be assigned to the stretching vibration band of –CH3 and –CH2 belonging to the alkyl groups and alkoxy groups in the COF skeletons. The solid-state 13C NMR spectra of these COFs exhibit the characteristic signal at 158 ppm for carbon in C=N bond, and the characteristic signals at 152, 149, 140, 132, 129, 119, and 115 ppm corresponding to the aromatic carbons in benzene ring and the carbons in porphyrin. The alkoxy carbon (–C–O–) and alkyl carbon (–C–C–) are observed at 56–72 and 11–31 ppm, respectively  spectroscopy, tic acid  M  and NaOH (1 M) solutions, obviously owing to the disassociation of imine linkages  facet. Other diffraction peaks in the PXRD pattern of DEG-HEP-COF at around 3.7°, 4.7°, 5.4°, 6.1°, 6.5°, 7.3°, and 7.9° are assignable to (020), (120), (200), (210), (220), (001), and (111) facets, respectively. The lattice mode of DEG-HEP-COF was simulated and the geometric energy of structures was minimized by the Material Studio. We noticed that the experimental PXRD pattern could not completely agree with the simulated pattern of conventional eclipsed AA or staggered AB stacking mode  and the experimental one (3.2°). According to the reported similar structure38, we allow the lattice distortion from regular square nets to the optimized conformation on the basis of the eclipsed AA stacking. As shown in Fig. a = 32.4 Å, b = 47.6 Å, c = 11.9 Å; α = β = γ = 90°) . In terms of the structure, the phenyl of the porphyrin and the terphenyl are twisted in contorted AA stacking mode. This phenomenon has been observed in other reported works about similar structures37. The steric hindrance of the chains of the adjacent layers prevents the formation of framework with a close-packed face-on orientation, and causes the formation of framework with a less closely twisted stacked conformation39. The flexibility of terphenyl moiety and the mutual attraction of side chains endow the bending and stacking of the skeleton with balance, which guarantee crystallinity of the COFs under the contorted stacking. The high-resolution transmission electron microscopy (HR-TEM) image shows highly ordered lattice fringes, confirming the periodically ordered pore structure of DEG-HEP-COF  to those of DEG-HEP-COF  measurement reveals significant differences in the morphology of these COFs  of DEG-HEP-COF with all alkyl chains or alkoxy groups being arranged in the same pore reveals that it is the most stable structure among all possible cases , including van der Waals dispersion, using Slater–Koster library in which O-N-H-C parameters were based on an early publicationses Fig. . Regardises Fig. . The calses Fig.  d. ThereWe further designed and synthesized the dialdehyde monomers with a single side chain  to construct the corresponding sCOFs  limits the combination of drugs49. The existing delivery systems loaded with hydrophilic and hydrophobic drugs have complex structures50. Considering the unique hetero-environmental porous structure of DEG-HEP-COF, we predicted it is a desired dual-antibiotic delivery vehicle. These synthesized COFs have no cytotoxicity to Huvecs and L929 cells by 3--2,5-diphenyltetrazolium bromide (MTT) assay  and 2% (w/v) antibiotic conditions) and low loading amounts for hydrophobic Lev  and 2% (w/v) antibiotic conditions). The HEP-COF shows the opposite trend  and 2% (w/v) antibiotic conditions, respectively)  infection was established to further evaluate the therapeutic effect of PG-DEG-HEP-COF@Antibiotics membranes as a potential wound dressing in vivo. The wound healing effect of the PG-DEG-HEP-COF@Lev&Van group was better than other treatment groups . In terms of the growth process, the experiment results indicate that the amphiphilic monomer possesses suitable crystallization rates. Meanwhile, theoretical calculation results suggest that the interaction of ethylene glycol chains exists in DEG-HEP-COF, which promotes the crystallization process, and the presence of alkyl chains regulates the crystallization rate and pore pattern to improve the crystallinity. The exploration of the growth process and the calculation indicate that the hydrophobic alkyl chain and hydrophilic alkoxy chain in the structure of DEG-HEP-COF are probably located in different pores. We further synthesized the sDEG-COF and sHEP-COF with the single side chain  to further indirectly verify the hetero-environmental pores structure from the pore size distribution. Furthermore, the DEG-HEP-COF exhibits low cytotoxicity and low hemolytic property, and can be utilized to load both hydrophobic and hydrophilic antibiotics for wound healing. This dual-antibiotic delivery strategy can expand the antibacterial range as compared with the single antibiotic ones, and delay or reduce the generation of drug resistance. In summary, the COF with hetero-environmental pores not only greatly expands the diversity of pore environments and manifests the significant influence of pore environments on the stacking mode and crystallinity but also provides a pathway for expanding the functionalities of COFs. The further exploration of COFs with multiple pore environments is in progress in our laboratory.In a typical procedure, the mixture of TAPP  and dialdehyde monomers (0.08 mmol), and mixture solvent with aqueous acetic acid (6 M)  mean statistically significant and ns means no significant difference. For all studies, samples were randomly assigned to various experimental groups.Data were represented as means ± SD. All data were obtained from the results of at least three independent experiments. Results are from different samples rather than repeated measurements of the same sample. Statistical comparisons of all data were analyzed by Excel 2020 , Origin 2018 , ImageJ , or IBM SPSS Statistics 25 . Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


Peptidoglycan amidases break the peptidoglycan layer during cell division and maintain integrity of the cell envelope. Here, we present structures of an isolated peptidoglycan hydrolase in an autoinhibited (“off”) state and a second amidase bound to the activating LytM domain of EnvC revealing the active (“on”) state. A comparison of these structures provides important molecular insights into the activation of peptidoglycan hydrolases by their cognate activators. Escherichia coli that are required to break the peptidoglycan layer during bacterial cell division and maintain integrity of the cell envelope. In vivo, the activity of AmiA and AmiB is tightly controlled through their interactions with the membrane-bound FtsEX–EnvC complex. Activation of AmiA and AmiB requires access to a groove in the amidase-activating LytM domain of EnvC which is gated by ATP-driven conformational changes in FtsEX–EnvC complex. Here, we present a high-resolution structure of the isolated AmiA protein, confirming that it is autoinhibited in the same manner as AmiB and AmiC, and a complex of the AmiB enzymatic domain bound to the activating EnvC LytM domain. In isolation, the active site of AmiA is blocked by an autoinhibitory helix that binds directly to the catalytic zinc and fills the volume expected to accommodate peptidoglycan binding. In the complex, binding of the EnvC LytM domain induces a conformational change that displaces the amidase autoinhibitory helix and reorganizes the active site for activity. Our structures, together with complementary mutagenesis work, defines the conformational changes required to activate AmiA and/or AmiB through their interaction with their cognate activator EnvC.AmiA and AmiB are peptidoglycan-hydrolyzing enzymes from  Escherichia coli (E. coli), splitting the peptidoglycan layer at the division site involves activity of three closely related periplasmic peptidoglycan amidases  have been identified including ActS  that blocks access to the amidase-binding groove in the EnvC LytM domain . Each AmiA monomer consists of a single globular domain with a six-membered beta sheet and six alpha helices . Both the experimental data and simulations consistently show that the regulatory domain is much more dynamic than the rest of the protein. The overall structure of AmiA is consistent with an autoinhibited form of the enzyme in which the active-site zinc is occluded by the blocking helix, while a potential protein interaction site remains exposed to solvent ready for activation.We determined a crystal structure of lae AmiB  and E. coli AmiC , includi helices A and B.  helices C. Simila helices  and AmiC helices , the Ami helices D. In oneE. coli. However, expression of AmiA variants that lack the regulatory domain causes a reduction in bacterial viability and increases bacterial sensitivity to detergent . This was the case for three distinct AmiA regulatory domain deletion constructs, each engineered with different regulatory domain deletions . In addition to the regulatory domain deletions, we also tested single-amino acid substitutions in Glu167 which is located on the blocking helix and, in the crystal structure, is directly ligated to the active-site zinc. The equivalent residue has previously been shown to be a key residue in maintaining autoinhibition for both AmiB . Molecular dynamics simulations of AmiA and AmiA Glu167 mutants provide useful context to these experiments, showing that the blocking helix fluctuates between bound and free positions in the mutants, but remains locked firmly in place for the wild type . These observations are consistent with a role for the blocking helix in AmiA autoinhibition, with Glu167 forming a “latch” that anchors the blocking helix to the active-site zinc.To test whether the regulatory domain maintains AmiA in an autoinhibited state, we made mutations that are predicted to relieve autoinhibition and monitored bacterial viability and detergent sensitivity when these variants were expressed in the periplasm. Expression of wild-type AmiA does not significantly disrupt viability or detergent sensitivity of oth AmiB  and AmiCSI Appendix, Fig. S5).We next turned our attention to the function of the interaction helix. Based on the structure of AmiA, we hypothesized that the solvent-facing hydrophobic residues presented along the face of the interaction helix mediate binding to the cognate activator (EnvC). Using a bacterial 2-hybrid experiment, we assessed the interaction between AmiA and the EnvC LytM domain after introducing point mutations into the interaction helix. Wild-type AmiA binds strongly to the EnvC LytM domain, but lysine substitutions of any of the solvent-facing hydrophobics completely disrupt the interaction A. When lSI Appendix, Fig. S6 A and B). Consistent with the bacterial 2-hybrid data, both AmiA L184K and L185K copurify in lower yield than the wild type, and the AmiA L188K variant does not interact at all, even though all AmiA variants are highly expressed in comparison to the LytM domain . We therefore conclude that the AmiA interacts with EnvC via its surface-exposed interaction helix.To further analyze the effect of interaction helix mutations in vitro, we coexpressed a subset of AmiA variants alongside the His-tagged EnvC LytM domain and assessed the stability of the complex using copurification . As expected from a previous triple-knockout study . We also showed that AmiA does not copurify with either the full-length EnvC protein or an EnvC construct lacking the coiled coil, consistent with EnvC being autoinhibited by the presence of the EnvC restraining arm in these constructs. These experiments complement previous work showing copurification of the EnvC periplasmic domain with AmiB, and bacterial 2-hybrid assays confirming this pattern of interactions for AmiA and AmiB in E. coli  that blocks access to the amidase-binding groove . This leE. coli EnvC (C. rodentium EnvC residues G319–G330) that contacts residues located between the interaction helix and helix 5. The extended loop wraps around the residues located between the interaction helix and helix 5, causing them to form a single, continuous, helical element . No sugars were used in crystallizing the complex and thus this molecule seems to have been copurified during protein production. The sugar could be a product of peptidoglycan hydrolysis; however, due to modest resolution (3.4 Å) and partial occupancy, we have not yet formally identified this molecule. We anticipate that future high-resolution studies may be able to resolve this ligand, and perhaps characterize further natural substrates, reaction products, or even inhibitors, bound to the amidase.Inspecting the density surrounding the zinc, a low-occupancy ligand is present at the fourth position of the coordination sphere. The ligand is consistent with a sugar molecule in chair conformation : AmiA is autoinhibited by its blocking helix which prevents the binding of peptidoglycan and the FtsEX–EnvC complex is autoinhibited by the restraining arm which prevents recruitment of the amidase to the LytM domain. Upon ATP binding to FtsEX–EnvC, a long-range conformational change is transmitted through EnvC, freeing the LytM domain from the restraining arm and exposing the amidase-binding site (Top Right). Binding of the amidase to the EnvC LytM domain relies upon the amidase interaction helix, which binds in the same location from which the restraining arm was displaced (Bottom). Upon binding, the amidase undergoes an induced conformational change in which the blocking helix is displaced from the active site. Rearrangement of the ligands surrounding the active-site zinc leads to peptidoglycan amidase activity. Finally, ATP hydrolysis allows the system to reset; the amidase is released and EnvC restraining arm returns to the LytM groove (Top Left). A near-identical mechanism likely operates for FtsEX–EnvC–AmiB, although in that case the amidase may additionally be prelocated at the division site by interactions between the N-terminal “AmiN” domain and the peptidoglycan layer  may well serve as a useful model for understanding these interactions.LytM domains are widespread among amidase activators , with prThe apparent redundancy between division-associated amidases and the large number of murein hydrolase activators raises the question of why such complexity is required. This is especially true for the FtsEX–EnvC–AmiA/AmiB system since the gram-positive equivalent, FtsEX–PcsB, operates without separate amidases and instead uses an EnvC-like protein (PcsB) that has its own peptidoglycan hydrolase activity , 32. MaiIn summary, we have determined the structures of two peptidoglycan amidases (AmiA and AmiB) in autoinhibited and activated states and relate these to their wider regulation through interactions with a cognate activator, the LytM domain of FtsEX–EnvC. The structures reveal near-atomic details of the conformational changes in the amidase that are required for activation of peptidoglycan hydrolysis including displacement of the autoinhibitory helix, and rearrangement of the sidechains that surround the active-site zinc. Our data significantly advance our understanding of a key event in bacterial cell division (breakage of the peptidoglycan layer) and provide fascinating molecular insights into the conformational changes that regulate amidase activity.SI Appendix. In brief, structures of AmiA and the complex between the AmiB enzymatic domain and the EnvC LytM domain were determined using X-ray crystallography using software from CCP4 suite (8C2O and 8C0J). Bacterial viability and detergent susceptibility were assessed by spotting out bacterial cultures in 10-fold series dilution on LB agar or LB agar supplemented with 0.1 % (w/v) SDS. All strains carry a plasmid providing ampicillin resistance as a selection marker and agar was supplemented with 50 μg/mL ampicillin and 1 mM IPTG. MICs were determined in microbroth culture using LB containing 50 μg/mL ampicillin and 1 mM IPTG. Bacterial 2-hybrid experiments used the BACTH system Click here for additional data file.


Based on the experimental data, the critical strain model and dynamic recrystallization model for the warm–hot forming of GCr15 bearing steel were established in this paper. The model accuracy is evaluated using statistical indicators such as the correlation coefficient (R). The dynamic recrystallization model exhibits high predictive accuracy, as indicated by an R-value of 0.986. The established dynamic recrystallization model for GCr15 bearing steel was integrated into the Forge® 3.2 numerical simulation software through secondary program development to simulate the compression process of GCr15 warm–hot forming. The dynamic recrystallization fraction was analyzed in various deformation regions. The grain size of the severe deformation zone, small deformation zone, and difficult deformation zone was compared based on simulated compression specimens under the conditions of 1050 °C and 0.1 s−1 with the corresponding grain size obtained with measurement based on metallographic photos; the relative error between the two is 5.75%. This verifies the accuracy of the established dynamic recrystallization and critical strain models for warm–hot deformation of GCr15 bearing steel. These models provide a theoretical basis for the finite element method analysis and microstructure control of the warm–hot forming process in bearing races.Warm deformation is a plastic-forming process that differs from traditional cold and hot forming techniques. At the macro level, it can effectively reduce the problem of high deformation resistance in cold deformation and improve the surface decarburization issues during the hot deformation process. Microscopically, it has significant advantages in controlling product structure, refining grain size, and enhancing product mechanical properties. The Gleeble-1500D thermal–mechanical physical simulation system was used to conduct isothermal compression tests on GCr15 bearing steel. The tests were conducted at temperatures of 600–1050 °C and strain rates of 0.01–5 s The ring rolling process is widely used to manufacture various seamless rings, especially in the production of bearing rings . Ring roGCr15 bearing rings typically operate under alternating tensile and compressive loads, and the microstructure and properties of the material can significantly impact its reliability and service life. Achieving a finer grain structure is critical to enhancing the material’s mechanical properties and service life ,10,11. I−1). They developed a dynamic recrystallization kinetics model based on an approximate stress–strain curve model. However, they did not combine their established dynamic recrystallization model with numerical simulation technology.The evolution of dynamic recrystallization is a dynamic process related to processing parameters. However, observing the dynamic evolution of the microstructure is extremely difficult. The numerical simulation gives an efficient approach to observing the evolution of the microstructure in GCr15 bearing steel . The finAt the moment, there are limited studies on the microstructure evolution and dynamic recrystallization model and microstructure numerical simulation of GCr15 bearing steel during warm forming.−1). The obtained metallographic photographs and rheological stress data from the compressed specimens were analyzed to investigate the microstructural evolution of GCr15. The accuracy of the dynamic recrystallization model for GCr15 was validated by comparing the grain size results obtained from finite element simulation with the actual grain size measurements from metallographic photographs. This provides a theoretical basis for predicting and controlling the grain size of bearing races during the microstructural evolution process in actual GCr15 bearing steel during warm–hot deformation.It is crucial to study the microstructure evolution of bearing rings and promote dynamic recrystallization degree during the forming process, which significantly improves the mechanical performance of bearing rings, such as strength, plastic toughness, and prolonging service life. Consequently, the GCr15′s characteristics of the microstructural evolution during warm–hot forming were studied in this article using numerical simulation techniques. The aim is to establish a dynamic recrystallization kinetics model for GCr15 that can effectively characterize the recrystallization evolution of the forming process. The specimens were undergoing compression tests using a Gleeble-1500D thermal–mechanical physical simulation system under varying temperatures and strain rates  with a maximum deformation of 50%.As illustrated in To analyze the microstructure of the compressed sample, it was axially cut and polished with varying grades of sandpaper and the metallographic polishing machine. After polishing, the sample was etched with picric acid and dodecylbenzene sulfonic acid aqueous solution and heated in a water bath at 80 °C for 3–5 min. After corrosion completion, the sample was cleaned, dried, and observed under an electron microscope to examine its microstructure.−1. In the initial stage of the compression test, the work hardening caused by the specimen’s deformation increases the true stress value sharply; upon reaching the critical strain, the true stress growth rate slows down, which can be attributed to the effects of the DRV and DRX mechanisms. As the deformation process continues, the dislocation density rapidly increases, leading to the further accumulation of strain energy. This increases the recrystallization driving force and deformation resistance, resulting in the peak stress at a true stress value of 113.812 MPa. With increasing true strain, there is a gradual decline in true stress. After a certain degree of deformation, DRX and DRV dominate, reducing the dislocation density and true stress values. With the further increase in true strain, the strengthening effect of work hardening and the softening effect of DRV and DRX tend towards dynamic equilibrium, stabilizing the true stress at 86.73 MPa.−1, 0.1 s−1, 1 s−1) are 98.943, 142.554, and 200.059 MPa, respectively. As the temperature increases, the level of thermal activation within the material becomes stronger, leading to an increase in the atomic diffusion rate. This results in more dislocations undergoing climb and cross-slip, and the number of slip systems increases, further enhancing the GCr15 steel’s ability to undergo plastic deformation, ultimately causing a decrease in the true stress values. On the other hand, recrystallization is predominantly a process of nucleation and grain growth. With increasing temperature, the driving force for dynamic recrystallization is enhanced.By observing As shown in −1) exhibited a higher degree of recrystallization than the specimen with the 5 s−1 strain rate. By observing −1. The experimental results demonstrated that decreasing the strain rate and elevating the processing temperature facilitate dynamic recrystallization.All specimens experienced varying degrees of DRX softening under different conditions. The true stress values decreased after reaching their peak stress. ®, making it a popular choice for engineers and researchers in the field.m1 and m9 define the material’s sensitivity to temperature, m5 term coupling temperature and strain, and m8 term coupling temperature and strain rate. m2, m4, and m7 define the material’s sensitivity to strain, m3 depends on the material’s sensitivity to the strain rate, T is the temperature given in Celsius.The “Hansel–Spittel” ,21 constThe model’s parameters were calculated using the averaging method ,23,24. AThe constitutive equation for GCr15 can be expressed as:Dynamic recrystallization refers to the procedure of grain nucleation and growth during metal deformation. Studying the microstructure evolution mechanism of DRX in GCr15 bearing steel during warm–hot deformation is crucial for analyzing its processability. Proposing an accurate theoretical model to describe the dynamic recrystallization process during the warm–hot forming process is essential for enhancing the precision of finite element simulation and predicting the microstructure evolution during deformation processes.As illustrated in The correlation between the critical strain and the peak strain has been demonstrated through studies :(3)εc=a⋅Poliak  proposedThe inflection point is the point of Therefore, the The extreme value point is the critical strain point:®, the result is shown in Equation (9), and the fitted curve is shown in The The parameters of the A linear regression of the critical and peak strains listed in n, and R is the gas constant. T is the absolute temperature. The peak strain can be calculated by using the Equation (10):Since the effect of the initial grain size on DRX is not investigated, it is assumed that the grain size is equal before deformation, i.e., it is constant, and Equation (10) can be simplified as:Taking the logarithm of the Equation (11):When the temperature is constant, The linear regression of the When the strain rate is constant, Linear regression was performed with least squares for The peak strain equation is:The critical strain equation is:, ,, and The Kopp model ,28,29 isLaasraoui et al. ,31 foundAs shown in ss point . Table 4m0 and C are constants; the value of θ can be expressed as θ is θ is 0, the value of When the stress value is between the critical stress and the saturation stress, the DRV curve can be expressed as:Integrating Equation (21), the boundary condition can be derived from After simplification, the dynamic recovery curve of the material during deformation is changed to Equation (23):The DRV curve can be obtained by substituting the values of The calculated dynamic recrystallization fraction under different deformation conditions is shown in −1. The figure shows numerous elongated and coarse-banded structures resulting from deformation. This indicates a high strain rate and a low degree of dynamic recrystallization. −1. With the increase in temperature, the degree of recrystallization increases, and some grains undergo the initial stage of dynamic recrystallization. The original grains’ grain borders are then invaded by new grains, forming a classic necklace-like arrangement. −1. Compared to −1. With increasing temperature, the driving force for recrystallization also increases, resulting in sufficient dynamic recrystallization. The grains exhibit a uniform size and are equiaxed in shape. The dynamic recrystallization characteristic strain is the strain at which dynamic recrystallization occurs in 50% of the internal volume of the material. The characteristic strain value of GCr15 steel can be obtained from Z parameter [Q is the thermal activation energy, R is the gas constant, and T is the absolute temperature. The arameter  can be rR is the gas constant. T is the absolute temperature.For the convenience of calculation, the Equation (24) can be rewritten as follows:Since the effect of the initial grain size on DRX is not investigated, it is assumed that the grain size is equal before deformation, i.e., it is constant, and the Equation (25) can be changed into:The coefficients of the characteristic strain model obtained from When the strain value reaches 50%, the recrystallization fraction is 0.5, and Equation (16) can be transformed into the following form:Therefore, it can be concluded that:The correlation between In summary, the dynamic recrystallization percentage model is:The Equation 30) can be used to calculate the DRX fraction of GCr15 steel for various deformation conditions.  can be u® numerical simulation software through secondary development. This will enable the software to perform warm compression simulations to validate the dynamic recrystallization model of GCr15 steel on cylindrical specimens.The Hansel–Spittle, dynamic recrystallization, and critical strain models developed for GCr15 steel will be integrated into the ForgeFinite element simulation is performed on 1/12 of the cylindrical sample, which is assumed to have cylindrical symmetry, for compression testing. The finite element simulation utilizes the Forge software from Transvalor, a company based in France. To ensure accuracy and convergence in the finite element calculation, tetrahedral elements are used to divide the parts. The upper and lower indenters are meshed with a size of 0.4 mm, while the cylindrical sample is meshed with a size of 0.15 mm. The thermal boundary conditions applied in the simulation are set to adiabatic conditions, similar to those used in the isothermal compression test. Other conditions are set to be consistent with the actual isothermal compression test.To validate the accuracy of the dynamic recrystallization models developed previously, a comparison was conducted between the numerical simulation results and the actual microstructure results from the compression tests.Due to friction between the specimen and die surface during the deformation process, the degree of deformation across different regions of the cylindrical sample varies. Region II is subject to lower frictional forces and shows a more uniform deformation. During the deformation process, it is under the stress state of “two-tensile-one-compress,” resulting in substantial deformation.Region III is the zone with the largest deformation. Despite the presence of strong three-directional compressive stress, it is located further away from the contact surface and is less obstructed during deformation. Hence, it is the most conducive area for slip and exhibits the greatest amount of deformation.According to ® 3.2)to simulate microstructure evolution.® software was used to validate the developed DRX model’s accuracy. The conclusions can be drawn as follows.The Gleeble-1500D testing machine was used in this paper to study the DRX behavior and microstructure evolution of GCr15 bearing steel in the process of warm–hot deformation. The microstructure dynamic recrystallization model and critical strain model of GCr15 under warm–hot deformation conditions were constructed, and the Forge(1)−1 to 5 s−1 and temperatures ranging from 600 °C to 1050 °C. This resulted in a decrease in true stress after attaining the maximum stress. The flow stress curve of the alloy shows three distinct phases. At the beginning of the test, the true stress increases sharply due to the work hardening resulting from the deformation of the specimen. Once the critical strain is reached, the rate of stress increase slows down because of the DRV and DRX mechanisms until it reaches the maximum stress. As the strain level rises, the true stress value declines until it reaches a steady-state stress level, where it stabilizes. On the other hand, at a specific strain rate, the maximum stress decreases as the deformation temperature elevates. Conversely, at a specific temperature, the maximum stress is proportional to the strain rate.The GCr15 specimens exhibited varying degrees of dynamic recrystallization softening under deformation conditions ranging from 0.01 s(2)The critical strain model of DRX during the warm–hot forming process of GCr15 was established using the work hardening rate method. The DRX kinetic model of GCr15 steel was developed with the stress–strain method. Under a fixed strain rate, the required strain to achieve the same DRX fraction decreases as the deformation temperature increases. However, at a constant temperature, it increases with an increase in strain rate.(3)® software, the established deformation dynamic recrystallization model, critical strain model, and Hansel–Spittle constitutive model for GCr15 steel were integrated into the software. The finite element simulation results suggest the distribution of the DRX fraction during the deformation of GCr15. The recrystallization fraction rises with a lower strain rate and a higher deformation temperature, which is completely consistent with the evolution law of the actual DRX volume fraction. In addition, a higher strain rate and a lower temperature can obtain a finer recrystallization grain structure.By performing secondary development on the Forge(4)The compressed specimens after isothermal compression present a bulging shape, and three typical deformation regions can be observed when the sample is cut along the axial direction. In the severe deformation region of the compressed sample, the deformation resistance is lower, resulting in a higher deformation amount and finer, more uniform grain size. The slight deformation region undergoes relatively uniform deformation due to minimal frictional effects. However, in the difficult deformation region, significant frictional effects make deformation more challenging, resulting in larger grain sizes in this area. Comparing the metallographic photos of the simulation results and the actual test results, it is found that the two have a high degree of agreement. It is further verified that the established related models have high accuracy and can provide a specific theoretical reference for actual production. It also shows that the finite element method (FEM) can provide an effective way for analyzing and predicting the microstructure evolution of the ring warm–hot deformation process.


Covalent organic frameworks (COFs) show enormous potential for building high-performance electrochemical sensors due to their high porosity, large specific surface areas, stable rigid topology, ordered structures, and tunable pore microenvironments.The basic properties, monomers, and general synthesis methods of COFs in the electroanalytical chemistry field are introduced, with special emphasis on their usages in the fabrication of chemical sensors, ions sensors, immunosensors, and aptasensors.The emerged COFs in the electrochemiluminescence realm are thoroughly covered along with their preliminary applications. Covalent organic frameworks (COFs), a rapidly developing category of crystalline conjugated organic polymers, possess highly ordered structures, large specific surface areas, stable chemical properties, and tunable pore microenvironments. Since the first report of boroxine/boronate ester-linked COFs in 2005, COFs have rapidly gained popularity, showing important application prospects in various fields, such as sensing, catalysis, separation, and energy storage. Among them, COFs-based electrochemical (EC) sensors with upgraded analytical performance are arousing extensive interest. In this review, therefore, we summarize the basic properties and the general synthesis methods of COFs used in the field of electroanalytical chemistry, with special emphasis on their usages in the fabrication of chemical sensors, ions sensors, immunosensors, and aptasensors. Notably, the emerged COFs in the electrochemiluminescence (ECL) realm are thoroughly covered along with their preliminary applications. Additionally, final conclusions on state-of-the-art COFs are provided in terms of EC and ECL sensors, as well as challenges and prospects for extending and improving the research and applications of COFs in electroanalytical chemistry. Since the discovery of boron-containing covalent organic frameworks (COFs) by Yaghi’s group in 2005 , this neElectrochemistry has been recognized as a versatile analytical tool over the past few decades and remains competitive in the face of growing demands in life analysis, environmental monitoring, and food inspection , 13. KnoIt is known that EC sensors rely most on multifarious electroactive or catalytic materials on the electrodes, even though the EC detection methods are much the same –25. ThusConsidering the great potential of the emerging COFs for building high-performance EC devices , this reThanks to several impressive reviews on the structural features and synthesis of COFs , 3, 5, dβ-ketoenamine, hydrazine, azine, and enamine . Inectively .Fig. 3ScBD-Tp (BD: benzidine) by a facile solution-phase method and loaded PtNPs by in-situ chemical reduction of H6PtCl4. After further integration of MWCNTs, the resultant ternary composites exhibited outstanding catalytic performance and electrical conductivity, affording effective drug sensing of tanshinol with a sensitivity up to 10.089 μA mM−1 and a LOD down to 0.018 μM arene (CP6)@COFPDA-Tp. The final heterogeneous composites of 2HP6@AuNPs@CP6@COFPDA-Tp with enhanced hydrophilicity were utilized for rapid EC monitoring of sodium picrate by virtue of the electrocatalysis of AuNPs, the recognition and enrichment of 2HP6 and CP6, as well as the outstanding supporting of COFPDA-Tp 3−/4− as the sensing indicator, a turn-off EC biosensor based on a flexible carbon paper electrode enabled the carbaryl detection with a low LOD of 0.16 μM . C. C3+/Fe20.031 μM .Thi-TFPB was synthesized from thionine (Thi) and TFPB via dehydration condensation and was further enwound by CNTs. With the improved stability and dispersibility, the COFs-supported CNTs exhibited prominent catalytic activity toward the oxidation of ascorbic acid (AA). Meanwhile, a pair of redox peaks assigned to electroactive COFThi-TFPB were inert to AA. As a result, a ratiometric strategy using the reference EC signal of COFThi-TFPB allowed for accurate AA sensing with a low LOD of 17.68 μM  trithiophene-2,5,8-tricarbaldehyde (BTT) and TTA were selected for building rod-like COFrichment . Also, Ce (BTDD) . The sam of HMIs .BTLP-1 on 3D-MC derived from kenaf stem was achieved by directly introducing 3D-MC into the amine-aldehyde polymerization process between 1,4-benzenedithiol-2,5-diamino-hydrochloride (BDDH) and TFB. The resultant COFBTLP-1/3D-MC composite contained regular transport channels and twelve adsorption sites for both the transfer and adsorption of HMIs, which was further exploited for simultaneous detection of HIMs .Fi.FiTAPB-Dπ clouds can promote charge/carrier transport. For example, Liu et al. demonstrated the use of PtNPs-decorated COF-LZU1 as an EC platform for immunoassay. Meanwhile, AuNPs/MOFs (HKUST-1) complexes containing massive Cu2+ ions served as EC probes to show the expression of C-reactive protein (CRP), an effective indicator of infection, with a low LOD of 0.2 ng mL−1 . . TAPB-DM fg mL−1 .2S2O8), a representative “reduction–oxidation” coreactant for ECL-active COFs, the ECL emitting route is described as the Reactions 2O82− is electro-reduced to produce the strong oxidant radical intermediate SO4·−. Meanwhile, ECL-active COFs undergo electro-reduction to form COFs·−. As a result, the radiative recombination of SO4·− and COFs·− generates high-energy COFs* to output ECL. To achieve high analytical sensitivity of ECL sensors, developing efficient ECL emitters and designing nanomaterials-based amplification strategies are the two main ways anilino]phenyl]-benzaldehyde (BCBA), respectively. In a similar manner, the D-A couples-powered IRCT aroused the ECL emitting out of these non-ECL active monomers. With endogenetic O2 as the coreactant, COFTBTN-BCBA achieved prominent ФECL of 63.7% in a water medium, which was further employed for accurate ECL monitoring of UO22+ with a proportionable ECL increase tetrabenzaldehyde (ETB), was utilized as a key monomer to prepare three olefin-linked COFs. These COFs exhibited predominant framework-induced ECL in aqueous solution, far exceeding those of monomeric aggregated states, because the ordered framework not only promoted efficient charge transfer for an excited-state generation but also reduced the ETB rotation-caused nonradiative loss [2O2. Further coupled with Co3O4 nanozyme for amplifying the signal, a molecularly imprinted polymer (MIP) ECL biosensor was constructed for antibiotic chloramphenicol testing with a LOD of 0.118 pM [sp2 carbon-conjugated COF NSs by C=C polycondensation between 2,2′- diacetonitrile (PDAN) and TFPPy. In this structure, the AIE-luminogens of cyano-substituted phenylenevinylene and the pyrene luminophores were topologically connected together to reduce the aggregation-caused quenching effect. Further using Bu4NPF6 as the coreaction accelerator of the COF NSs/S2O82− ECL system, an ECL genosensor enabled the microRNA-21 detection with a low LOD of 46 aM via three cascaded Exo III-mediated recycling processes for signal amplification [AIE refers to the fact that a material emits intense luminescence in the aggregated state, but no or weak light in the solution state , 136. Deive loss . Li et a0.118 pM . In anot0.118 pM . Zhang efication .−8 S m−1). To address this issue, Zhang et al. designed a conductive COF with a fully π-conjugated planar structure by connecting HHTP and 2,3,6,7,10,11-hexaaminotriphenylene (HATP) via pyrazine linkages. The splendid ECL emission was achieved as a result of the superior electronic conductivity (3.11 × 10−4 S m−1), the luminophores directly as building blocks, and the porous structure that allows free diffusion of electrolyte. Based on COFHHTP-HATP after prereduction electrolysis in S2O82− solution as an efficient solid-state ECL material, an “off–on” ECL platform was established for sensitive determination of thrombin through aptamer/target proximity binding-mediated 3D bipedal DNA walker for signal amplification [π conjugated COFDMTP-TCPB with sp2 carbon-linkage via Knoevenagel polycondensation of DMTP and 1,3,5-tris(4-cynomethylphenyl)benzene (TCPB), exhibiting high ECL efficiency for alkaline phosphatase detection [Although COFs have a high assembling capacity for ECL phosphors or motifs, the intrinsic poor conductivity still limits the ECL performance , as a building motif. Owing to the topologically ordered and porous architectures, this Ru-MOF displayed strong ECL emitting and remarkable chemical stability for the microRNA-155 detection, achieving an ultralow LOD of 3.02 aM [2 NSs into Mn2+, restoring the ECL emission accordingly [22+ [Additionally, various ECL-active COFs are springing up, accompanying their ECL sensing applications. For instance, Yang et al. designed a stable Ru-complex-based metal-COF using a classic ECL luminophore, trisruthenium(II) (Ru(dcbpy) 3.02 aM . Song et 3.02 aM . The samordingly . Lately,ordingly . This grgly [22+ . Besidesgly [22+ .32+) onto COF-LZU1, displaying increased nearly fivefold ECL intensity with a standard of classical Ru(bpy)32+/tripropylamine (TPrA) system. In this work, COF-LZU1 can not only load massive Ru(bpy)32+ but also enrich a large amount of TPrA from the solution via the hydrophobic interaction. Thus, using COF-LZU1 as an ECL microreactor, a confined microenvironment was well created for the redox and survival of coreactants. Through Nt. BbvCI restriction endonuclease-powered DNA walker and T7 exonuclease-mediated target recycling for dual signal amplification, these microreactors were further employed for ultrasensitive ECL detection of aflatoxin M1, showing a low LOD of 0.009 pg mL−1 [In addition to being directly used as ECL emitters, COFs can also be made into microreactors to accumulate luminophores and coreactants, thus boosting ECL efficiency due to the confinement effect. For example, Zeng et al. loaded tris ruthenium(II) (Ru(bpy) pg mL−1 .π–π interactions [3N4 nanosphere -N-ethylisoluminol (ABEI) was also linked to COF-LZU1 as an ECL emitter for label-free signal-off detection of cytochrome c. This sensor exhibited a wide linear range of 1.00 fg mL−1–0.10 ng mL−1 and a low LOD down to 0.73 fg mL−1 [In addition, COFs with high specific surface area and porosity can also serve as excellent electrode matrixes to support catalytic centers in a well-dispersed manner for amplified ECL outputs. For example, a liquid–liquid interface assembly strategy allowed for the polymerization of Zr-coordinated amide porphyrin-based COFs. This Zr-porphyrin-based COF adopted a 2D multilayer structure with high conductivity and electrocatalysis performance toward the luminol/Hl effect . Li and o 7.9 pM . Ma et a 2.21 fM . Besides fg mL−1 .π-stacked or extended π-conjugated backbones, for example, that are COFs via pyrazine or olefin linkage, possess acceptable electrical conductivity. (6) Ratiometric EC sensing can be executed for accurate results based on the unique electroactive COFs.This review primarily provides an overview of the state-of-the-art COFs utilized in the field of electroanalytical chemistry, sketching the trajectory from basic characteristics and general synthesis strategies to the resultant sensing usages. The progress of elaborate COFs-based EC sensors is discussed systematically, including ions sensors, chemical sensors, immunosensors, and aptasensors. These COFs-based EC sensors demonstrated superior analytical performance. (1) Until now, imine-linked COFs formed by aminaldehyde condensation remain the most reported COFs for EC sensing applications due to their facile synthesis, high crystallinity, and stability. (2) Their high specific surface area provides a large electrode active area and high loading capacity, and the super-micropores and ordered channels ensure favorable energy/mass transfer. (3) Nano-sized pores and existing heteroatoms can trap guest targets through non-covalent interactions. (4) COFs generally exhibit flexibility, good biocompatibility, low toxicity, and high stability, beneficial for the fabrication of repeatable and reproducible EC sensors. (5) Framework networks designed by π-conjugated planar structure can improve the conductivity and achieve fast charge injection. (4) The inherent characteristics of non-ECL active COFs make them the right microreactors, strong absorbents, and electrode matrices with high specific surface area, thereby improving the performance of ECL sensors.In addition, ECL available COFs and their preliminary sensing applications are summarized in detail. (1) At present, modulating the IRCT behavior of COFs is a common strategy to activate ECL from non-ECL active monomers. (2) AIE-ECL COFs can be facilely achieved because the rigid framework restricts the intramolecular rotations and vibrations of luminogens. (3) Designing COFs with a fully Therefore, we believe that COFs are capable of taking a place in the field of electroanalytical chemistry, accompanied by the development of advanced synthetic methods and the disclosure of unique attributes in future work. Despite impressive progress, various obstacles remain to be addressed for the wide applications of COFs in electroanalytical chemistry. First, the solvothermal method still dominates the COFs preparation, but it usually requires tedious synthesis procedures and harsh reaction conditions. Second, COFs composed of non-conductive organic motifs are severely restricted as electroactive substances for EC sensing. The synthesis of highly conductive COFs is still relatively challenging work. Third, due to poor affinity, the assembly of COFs at the interface makes it difficult to achieve high efficiency and reproducibility. Most of the current works focus on the direct use of COFs while ignoring the vital role of modification strategies. Fourth, theoretical prediction of the covalent ordered structure of COFs and their EC behaviors is an arduous task. Lastly, the potential biotoxicity of COFs may affect their further commercial applications.π-conjugated planar structures. Assembling COFs-derived composites with particular architecture can also access good conductivity and affinity. Usually, COFs prepared through the traditional solvothermal method exhibit nonuniform bulky morphology, so it is meaningful to develop synthetic strategies for special structures, such as hollow nanostructures mimicking containers, vesicles, and cells. Certainly, developing facile, green, and mild synthetic methods is a must for the large-scale manufacture of COFs-based EC sensors. Pre-functionalized monomers or post-synthetic modifications to exploit the diversity of species and functions of COFs have great potential for future development. Furthermore, most reported COFs possess only micropores, preventing guests  from entering the active cavities or open channels, and creating hierarchical porosity in crystal COFs is highly desired accordingly. In particular, the in-situ interface assembly strategy of COFs awaits further exploration to establish stable and repeatable sensing platforms. In the ECL domain, although some ECL available COFs have been discovered, the ECL research on COFs is still in its initial stage. Future research is suggested to focus on the design of novel COFs emitters, the study of structure-related ECL behaviors, the dependence of their physicochemical properties and electronic structure on the ECL efficiency, the disclosure of underlying ECL reactions occurring in COFs, as well as expanding ECL assays.A few shallow outlooks are only for the reference of readers. In terms of the EC sensing aspect, it is a promising direction to develop conductive COFs with completely