FoxO induces pupal diapause by decreasing TGFß signaling.

Diapause is a form of dormancy used widely by insects to survive adverse seasons. Previous studies have demonstrated that forkhead box O (FoxO) is activated during pupal diapause initiation in the moth Helicoverpa armigera. However, it is unclear how FoxO induces diapause. Here, we show that knockout of FoxO causes H. armigera diapause-destined pupae to channel into nondiapause, indicating that FoxO is a master regulator that induces insect diapause. FoxO activates the ubiquitin-proteasome system (UPS) by promoting ubiquitin c (Ubc) expression via directly binding to the Ubc promoter. Activated UPS decreases transforming growth factor beta (TGFß) receptor signaling via ubiquitination to block developmental signaling to induce diapause. This study significantly advances the understanding of insect diapause by uncovering the detailed molecular mechanism of FoxO.

Programmable DNA Scaffolds Enable Orthogonal Engineering of Cell Membrane-Based Nanovesicles for Therapeutic Development.

Cell membrane-based nanovesicles (CMNVs) play pivotal roles in biomolecular transportation in living organisms and appear as attractive bioinformed nanomaterials for theranostic applications. However, the current surface-engineering technologies are limited in flexibility and orthogonality, making it challenging to simultaneously display multiple different ligands on the CMNV surface in a precisely controlled manner. Here, we developed a DNA scaffold-programmed approach to orthogonally engineer CMNVs with versatile ligands. The designed DNA scaffolds can rapidly anchor onto the CMNV surface, and their unique sequences and hybridized properties enable independent control of the loading of multiple different types of biomolecules on the CMNVs. As a result, the orthogonal engineering of CMNVs with a renal targeted peptide and a therapeutic protein at controlled ratios demonstrated an enhanced renal targeting and repair potential in vivo. This study highlights that a DNA scaffold-programmed platform can provide a potent means for orthogonal and flexible surface engineering of CMNVs for diverse therapeutic purposes.

Multifunctional DNA-Based Hydrogel Promotes Diabetic Alveolar Bone Defect Reconstruction.

Diabetic alveolar bone defect (DABD) causes persistent bacterial infection, prolonged inflammation, and delayed bone healing, making it a considerable clinical challenge. In this study, by integrating silver nanoclusters (AgNCs) and M2 macrophage-derived extracellular vesicles (M2EVs), a multifunctional DNA-based hydrogel, called Agevgel, is developed with antibacterial, anti-inflammatory, immunomodulatory, and osteogenic properties to promote DABD rebuilding. AgNCs are tightly embedded into the DNA scaffolds and exhibit effective anti-bacterial activity, while immunomodulatory M2EVs are encapsulated within the shape-variable DNA scaffolds and exhibit potent anti-inflammatory and osteogenic properties. The results reveal that Agevgel effectively prolongs the local retention time and bioactivity of M2EVs in vivo. In particular, the sustained release of M2EVs can last for at least 7 days when applying Agevgel to DABD. Compared to free M2EVs or Aggel (AgNCs encapsulated within the DNA hydrogel) treatments, the Agevgel treatment accelerates the defect healing rate of alveolar bone and dramatically improves the trabecular architecture. Mechanistically, Agevgel plays a key role in regulating macrophage polarization and promoting the expression of proliferative and osteogenic factors. In summary, Agevgel provides a comprehensive treatment strategy for DABD with a great clinical translational value, highlighting the application of DNA hydrogels as an ideal bioscaffolds for periodontal diseases.

Investigation into fabrication and optical characteristics of tunable optofluidic microlenses using two-photon polymerization.

Optofluidic systems, integrating microfluidic and micro-optical technologies, have emerged as transformative tools for various applications, from molecular detection to flow cytometry. However, existing optofluidic microlenses often rely on external forces for tunability, hindering seamless integration into systems. This work presents an approach using two-photon polymerization (TPP) to fabricate inherently tunable microlens arrays, eliminating the need for supplementary equipment. The optofluidic design incorporates a three-layered structure enabling dynamic manipulation of refractive indices within microchannels, leading to tunable focusing characteristics. It is shown that the TPP fabricated optofluidic microlenses exhibit inherent tunable focal lengths, numerical apertures, and spot sizes without reliance on external forces. This work signifies some advancements in optofluidic technology, offering precise and tunable microlenses with potential applications in adaptive imaging and variable focal length microscopy.

I2-DMSO-Mediated Construction of 2,3- and 2,4-Disubstituted Pyrimido[1,2-b]indazole Skeletons.

An efficient synthetic method for constructing 2,3- and 2,4-disubstituted pyrimidio[1,2-b]indazole skeletons through I2-DMSO-mediated and substrate-controlled regioselective [4 + 2] cyclization is reported. The reaction conditions are mild, its operation is simple, and the substrate scope is wide. More than 60 pyrimidio[1,2-b]indazole derivatives have been synthesized, providing a new methodology for constructing related molecules and potentially enriching bioactive-molecule libraries.

TRIM21 Regulates Virus-Induced Cell Pyroptosis through Polyubiquitination of ISG12a.

Pyroptosis is a form of regulated cell death mediated by the gasdermin protein family. During virus infection, cell pyroptosis restricts viral replication. The mechanisms of the tripartite motif (TRIM) protein family and IFN-stimulated genes (ISGs) against viruses have been studied. The role of TRIMs and ISGs in pyroptosis remains unclear. In this study, we show that TRIM21 interacts with ISG12a in viral infection and facilitates its translocation into the mitochondria by promoting its ubiquitination, thereby causing caspase 3 activation. Gasdermin E (GSDME) is specifically cleaved by caspase 3 upon viral infection, releasing the GSDME N-terminal domain, perforating the cell membrane, and causing cell pyroptosis. Our study uncovers a new mechanism of TRIM21 and ISG12a in regulating virus-induced cell pyroptosis.

A Directly Moldable, Highly Compact, and Easy-for-Integration 3D Micromixer with Extraordinary Mixing Performance.

Micromixers are a critical component in microfluidics. However, most 2D passive micromixers produce optimal mixing at a high flow rate range and 3D micromixers require mm-scale channels or a complex assembly that is unsuitable for microfluidic applications. Here, we reported a 3D PDMS micromixer based on the splitting-stretching-recombination (SSR) of streams to facilitate molecular diffusion, which can effectively and rapidly mix solutions with low Reynolds numbers (0.01-10). The fabrication of our micromixer is convenient with only two steps─two-photon polymerization (2PP) 3D printing and soft lithography, with high resolution, reproducibility, and ease for integration. We investigated the mixing performance of the micromixer by CFD simulations and experimental studies under a confocal microscope; the results confirmed its better performance and higher chip miniaturization than others. It can achieve a mixing efficiency above 0.90 (which is generally regarded as complete mixing) for low-Re solutions (flow rates ≤60 µL/min) with a mixing volume smaller than 20 nL. The time for complete mixing is in the range of milliseconds (e.g., 21 ms for Re = 10, 194 ms for Re = 0.88). The device shows negligible degradation in mixing performance for highly viscous solutions (∼50 times more viscous than water), macromolecule solutions, and colloidal solutions of nanoparticles.

Femtosecond laser multibeam parallel processing for variable focal-length optofluidic chips.

Optofluidic chips are frequently utilized in applications such as biological observation, chemical detection, dynamic displays, imaging, holography, and sensing. Yet, developing continuously zoomable technology has been challenging in the production of optical devices. Using a spatial light modulator to shape a femtosecond laser to achieve multibeam parallel pulse punching, we propose an easy-to-fabricate, stable, and reliable tuning technique in this Letter. We then propose the addition of a liquid medium with a continuously variable refractive index to achieve controllable zooming without changing the position and morphology of the microlens. By pumping various concentrations of the liquid medium into the optofluidic chip, continuous tunability of the device was experimentally verified.

Removal effect of trihalomethanes (THMs) and halogenated acetic acids (HAAs) precursors in reclaimed water by polyaluminum chloride (PACl) coagulation.

This study analyzed the removal effect of various doses of polyaluminum chloride (PACI) on wastewater treatment plants at pH 7. The sewage plant's secondary effluent organic matter (EfOM) separates into four components: hydrophobic base (HOB), hydrophilic (HI), hydrophobic acid (HOA), and hydrophobic neutral (HON). The removal effect for various forms of organic waste is optimum at 16 mg/L and that halogenated acetic acids (HAAs) and trihalomethanes (THMs) are formed simultaneously. After PACI treatment, hydrophobic organic compounds were converted to humic acid (HA), fulvic acid (FA), soluble microbial products (SMPs), and other HI organic compounds, increasing the amount of HAAs produced by HI fractions. Removal rate of hydrophobic organic compounds, particularly HON, is 92.8% when using PAC. Moreover, after EfOM coagulation, most HAAs are trichloroacetic acid (TCAA), followed by bromochloroacetic acid (BCAA) and bromodichloroacetic acid (BDCAA). Only HOB can produce monochloroacetic acid (MCAA), whereas HA and SMPs with HOA are primary components of dichloroacetic acid (DCAA). The toughest removable byproduct of THMs is CHBr3, and after condensation of each THM component, only HOA and HON produce CHBr3, while HI produces only a minimal quantity of CHBrCl2 and CHCl3.This finding is critical for understanding how disinfection byproducts are produced after chlorinating EfOM.

Adiponectin gene therapy prevents islet loss after transplantation.

Significant pancreatic islet dysfunction and loss shortly after transplantation to the liver limit the widespread implementation of this procedure in the clinic. Nonimmune factors such as reactive oxygen species and inflammation have been considered as the primary driving force for graft failure. The adipokine adiponectin plays potent roles against inflammation and oxidative stress. Previous studies have demonstrated that systemic administration of adiponectin significantly prevented islet loss and enhanced islet function at post-transplantation period. In vitro studies indicate that adiponectin protects islets from hypoxia/reoxygenation injury, oxidative stress as well as TNF-α-induced injury. By applying adenovirus mediated transfection, we now engineered islet cells to express exogenous adiponectin gene prior to islet transplantation. Adenovirus-mediated adiponectin transfer to a syngeneic suboptimal islet graft transplanted under kidney capsule markedly prevented inflammation, preserved islet graft mass and improved islet transplant outcomes. These results suggest that adenovirus-mediated adiponectin gene therapy would be a beneficial clinical engineering approach for islet preservation in islet transplantation.

TRIM10 binds to IFN-α/ß receptor 1 to negatively regulate type I IFN signal transduction.

The type I interferon (IFN-I) system is important for antiviral and anticancer immunity. Prolonged activation of IFN/JAK/STAT signaling is closely associated with autoimmune diseases. TRIM10 dysfunction may be associated closely with certain autoimmune disorders. Here, we observed that the serum TRIM10 protein level is lower in patients with systemic lupus erythematosus than in healthy control subjects. We speculated the possible involvement of TRIM10-induced modulation of the IFN/JAK/STAT signaling pathway in systemic lupus erythematosus. In line with our hypothesis, TRIM10 inhibited the activation of JAK/STAT signaling pathway triggered by various stimuli. TRIM10 restricted the IFN-I/JAK/STAT signaling pathway, which was independent of its E3 ligase activity. Mechanistically, TRIM10 interacted with the intracellular domain of IFNAR1 and blocked the association of IFNAR1 with TYK2. These data suggest the possible TRIM10 suppresses IFN/JAK/STAT signaling pathway through blocking the interaction between IFNAR1 and TYK2. Targeting TRIM10 is a potential strategy for treating autoimmune diseases.

A study on the accuracy of a new fluorescent detection method for vaginal fungi.

BACKGROUND: To investigate the positive rate and clinical applicability of liquid-based fungal method for detecting of vaginal fungi. We collect the secretions from the posterior vaginal fornix and the vaginal wall of 198 patients with clinically suspected fungi vaginitis patients for study. METHODS: The vaginal fungi of vaginal discharge were detected by fluorescence method, i.e., by liquid-based thin-layer fungi fluorescence morphology staining detection kit (liquid-based fungal method), saline smear method and fungal culture method. RESULTS: The positive rate of liquid-based fungal method, saline smear method was 50%, 25.75% respectively. The positive rate of liquid-based fungal method were 50%. The true positive rate of liquid-based fungal method (87.85%) was higher than that of saline smear method (45.79%, P < 0.001), which was easy to miss diagnosis. Moreover, the Kappa (K) of liquid-based fungal method was 0.81, and P < 0.01, which was statistically significant, indicating that the consistency of the two detection methods is good. Of the eight common symptoms of fungal vaginitis, the positive symptom coincidence rate of liquid-based fungal method was consistent with that of fungal culture method. It was also easier to see fungi under a microscope than with saline smear method. CONCLUSION: The liquid-based fungal method has a high positive coincidence rate and accuracy in the detection of vaginal fungi, and it is convenient to operate and implement steps. Therefore, it may be applied in clinical practice. Or a combination of several detection methods can be used.

Identification of MDM2, YTHDF2 and DDX21 as potential biomarkers and targets for treatment of type 2 diabetes.

Type 2 diabetes (T2D) is a multifactorial and polygenetic disease, although its exact etiology remains poorly understood. The objective of this study was to identify key biomarkers and potential molecular mechanisms in the development of T2D. Human RNA-Seq datasets across different tissues (GSE18732, GSE41762, and GSE78721) were collected from the Gene Expression Omnibus (GEO) database and differentially expressed genes (DEGs) between T2D and controls were identified using differential analysis. A total of 90 overlapping DEGs were identified, among which YTHDF2, DDX21, and MDM2 were considered as key genes due to their central positions in the PPI network and the same regulatory pattern in T2D. Logistic regression analysis showed that low expression of the key genes increased the risk of T2D. Enrichment analysis revealed that the key genes are involved in various important biological functions and signaling pathways including Notch, Fork head box O (FOXO), and phosphoinositide 3-kinase (PI3K)-Akt. RT-qPCR and Western blot analysis showed that all three key genes were down-regulated in pancreatic islets of both prediabetic and diabetic mouse models. Finally, the insulin-sensitizer, pioglitazone was used to treat db/db mice and immunofluorescence analysis showed that the expression of all three key genes was significantly down-regulated in db/db islets, an effect that was overcome by pioglitazone treatment. Together, these results suggest that the identified key genes could be involved in the development of T2D and serve as potential biomarkers and therapeutic targets for this disease.

Tolvaptan treatment of cystine urolithiasis in a mouse model of cystinuria.

INTRODUCTION: Cystinuria is an inherited disease characterized by increased urinary cystine excretion and recurrent cystine stones. Current treatment regimens have limited effectiveness in preventing stone recurrence and are often poorly tolerated. The aim of this study was to evaluate the effect of tolvaptan, a vasopressin receptor 2 (V2) antagonist, on cystine stone volume in mice with cystinuria. MATERIALS AND METHODS: Tolvaptan (0.4 mg per mouse) or placebo was delivered by gavage daily for 30 days. Urinary amino acids and cystine stones were analyzed to assess drug efficacy in preventing L-cystine stone growth using several analytical methods. Data were entered into SPSS and analyzed by paired sample T test. p value < 0.05 was considered significant. RESULTS: Compared with control group, the liquid intake and urine volume in tolvaptan-treated mice were significantly increased. The urinary cystine concentrations in group tolvaptan was lower than the baseline concentration before the experiment. After treatment, mice treated with tolvaptan had significantly delayed stone growth, exhibited lower overall stone volume accumulation, compared with control group. The increased stone volume of tolvaptan group was less than control group (8.00 ± 4.93 mm3 vs 27.90 ± 4.48 mm3, p < 0.001). The serum creatinine in the control group (11.75 ± 1.634 µmol/L) was higher than that in the tolvaptan group (7.625 ± 1.401 µmol/L) (p = 0.0759). In addition, tolvaptan significantly inhibited the formation and growth of stones in mice after cystolithotomy. CONCLUSION: The present study indicated that tolvaptan's efficacy in preventing L-cystine stone growth through increased liquid intake and urine volume of cystinuric mice.

Comparison of vacuum-assisted sheaths and normal sheaths in minimally invasive percutaneous nephrolithotomy: a systematic review and meta-analysis.

BACKGROUND: A systematic review and meta-analysis was conducted to compare the safety and efficacy of vacuum-assisted sheaths and conventional sheaths in minimally invasive percutaneous nephrolithotomy (MPCNL) in the treatment of nephrolithiasis. METHODS: PubMed, Web of Science, Embase, EBSCO, and Cochrane Library databases (updated March 2021) were used to search for studies assessing the effect of vacuum-assisted sheaths in patients who underwent MPCNL. The search strategy and study selection processes were implemented in accordance with the PRISMA statement. RESULT: Three randomized controlled trials and two case-controlled trials that satisfied the inclusion criteria were enrolled in this meta-analysis. Overall, the stone-free rate (SFR) in patients who underwent vacuum-assisted sheaths was significantly higher than that in patients who underwent conventional sheaths (RR 1.23, 95% CI 1.04, 1.46, P = 0.02), with significant heterogeneity among the studies (I2 = 72%, P = 0.03). In terms of the outcome of complications, vacuum-assisted sheath could bring a benefit to the postoperative infection rate (RR 0.48, 95% CI 0.33, 0.70, P < 0.00001) with insignificant heterogeneity among the studies (I2 = 0%, P = 0.68). There was no significant difference in the blood transfusion rate (RR 0.35, 95% CI 0.07, 1.73, P = 0.17), with significant heterogeneity (I2 = 66%, P = 0.35). Three studies contained operative time data, and the results indicated that the vacuum-assisted sheath led to a shorter operative time (MD = - 15.74; 95% CI - 1944, - 12.04, P < 0.00001) with insignificant heterogeneity (I2 = 0%, P = 0.91). CONCLUSION: The application of a vacuum-assisted sheath in MPCNL improves the safety and efficiency compared to the conventional sheath. A vacuum-assisted sheath significantly increases the SFR while reducing operative time and postoperative infection.

Comparison of nephroscopy and cystoscopy used in the treatment of bladder stones: a systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: A systematic review and meta-analysis was conducted to compare the safety and efficiency of nephroscopy and cystoscopy in transurethral cystolithotripsy (TUCL) for bladder stones (BS). METHODS: The PubMed, Web of Science, Embase, EBSCO, and Cochrane Library databases were searched up to January 2021 for studies assessing the effect of different types of endoscopes among patients who underwent TUCL. The search strategy and study selection process were in accordance with the PRISMA statement. RESULTS: Five randomized controlled trials were included in the meta-analysis. The results showed no difference in stone-free rate (RR = 1.00, CI = 0.98-1.02, p = 1.00) between the two groups and nonsignificant heterogeneity (I2 = 0%, p = 1.00), and all patients were rendered stone free. Use of the nephroscope significantly shortened the operative time compared with the cystoscope group (RR= - 26.26, CI = - 35.84 to - 16.68, p < 0.00001), and there was significant heterogeneity (I2= 87%, p < 0.00001). There was no significant difference in mean urethral entries (RR = 0.66, CI = - 0.71 to - 2.04, p = 0.35), hospitalization (MD = 0.08, 95% CI = - 0.07 to 0.23, p = 0.31) or total complication rate (RR=1.37, 95% CI = 0.47-4.00, p = 0.56) between the two groups. CONCLUSIONS: In conclusion, this systematic review demonstrates that both nephroscopy and cystoscopy have high stone clearance efficiency, low rates of complications and short hospitalizations. The mean urethral entries depend on the treatment method for large stone fragments. However, the use of nephroscopy can significantly reduce the operative time.

Small Object Detection in Traffic Scenes Based on Attention Feature Fusion.

There are many small objects in traffic scenes, but due to their low resolution and limited information, their detection is still a challenge. Small object detection is very important for the understanding of traffic scene environments. To improve the detection accuracy of small objects in traffic scenes, we propose a small object detection method in traffic scenes based on attention feature fusion. First, a multi-scale channel attention block (MS-CAB) is designed, which uses local and global scales to aggregate the effective information of the feature maps. Based on this block, an attention feature fusion block (AFFB) is proposed, which can better integrate contextual information from different layers. Finally, the AFFB is used to replace the linear fusion module in the object detection network and obtain the final network structure. The experimental results show that, compared to the benchmark model YOLOv5s, this method has achieved a higher mean Average Precison (mAP) under the premise of ensuring real-time performance. It increases the mAP of all objects by 0.9 percentage points on the validation set of the traffic scene dataset BDD100K, and at the same time, increases the mAP of small objects by 3.5%.

Novel nomograms for castration-resistant prostate cancer and survival outcome in patients with de novo bone metastatic prostate cancer.

OBJECTIVES: To develop nomograms predicting the incidence of castration-resistant prostate cancer (CRPC) and overall survival (OS) for de novo metastatic prostate cancer (PCa). PATIENTS AND METHODS: Data from 449 patients with de novo metastatic PCa were retrospectively analysed. Patients were randomly divided into a training (n = 314, 70%) and a validation cohort (n = 135, 30%). Predictive factors were selected using a Cox proportional hazards model and were further used for building predictive models. The outcomes were incidence of CRPC and OS. RESULTS: Predictive factors included: Gleason score (GS), intraductal carcinoma of the prostate (IDC-P), Eastern Cooperative Oncology Group status, and alkaline phosphatase, haemoglobin and prostate-specific antigen levels. IDC-P and GS were the strongest prognosticators for both the incidence of CRPC and OS. Nomograms for predicting CRPC and OS had an internal validated concordance index of 0.762 and 0.723, respectively. Based on the ß coefficients of the final model, risk classification systems were constructed. For those with favourable, intermediate and poor prognosis, the median time to CRPC was 62.6, 28.0 and 13.0 months (P < 0.001), respectively; and the median OS was not reached, 55.0 and 33.0 months, respectively (P < 0.001). CONCLUSIONS: We developed two novel nomograms to predict the incidence of CRPC and OS for patients with de novo metastatic PCa. These tools may assist in physician decision-making and the designing of clinical trials.

Six-Part Classification of Femoral Intertrochanteric Fractures: A Classification Method to Improve the Diagnosis Rate of Unstable Fractures.

OBJECTIVE: Three-dimensional (3D)-CT data is currently insufficient for classifying femoral trochanter fractures. Fracture classification based on fracture stability analysis is helpful to evaluate the prognosis of patients after internal fixation. Currently, there is a lack of fracture classification methods based on 3D-CT images and fracture stability analysis. The aim of this study was to propose a new six-part classification method for intertrochanteric fractures of femur based on 3D-CT images and fracture stability analysis to improve the diagnosis rate of unstable fractures. METHOD: From January 2009 to December 2019, 320 patients receiving intramedullary nail surgery for femoral intertrochanteric fractures at Chengdu University's Affiliated Hospital were studied retrospectively. AO and six-part classifications were undertaken according to the 3D-CT image data of the patients, and the stability rates of two classifications were compared. According to the six-part classification stability criteria, the patients were divided into a stable and an unstable fracture group. The perioperative and follow-up indicators of the two groups were statistically analyzed, and the six-part classification's inter-observer and internal reliability was examined. RESULTS: There were 107 men and 213 females women the 320 patients, with an average age of 79.32 ± 11.26 years and an osteoporosis rate of 55.63% (178/320). The fracture stability rate of 39.69% (127/320) was studied using a six-part classification method. The AO classification fracture stability rate was 42.50% (136/320), with no significant difference (χ2 = 0.523, p = 0.470 > 0.05). There is no statistically significant difference between the two classification techniques in the examination of fracture stability (McNemer difference test p = 0.306 > 0.05; Kappa consistency test p < 0.001). According to the six-part classification, fracture stability and instability group were divided into two groups. The following indicators were compared between the two groups: The surgery time (p = 0.280), fracture reduction quality (p = 0.062); function independent measurement (p = 0.075); timed up and go test (TUG) (p = 0.191), and Parker-Palmer score (p = 0.146). Were as compared according to the six-part classification of stable and unstable fracture groups. Perioperative blood loss (p < 0.001), the Harris score excellent and good rate (p = 0.043), fracture healing time (p < 0.001), and the entire weight-bearing duration (p = 0.002) were statistically significant. The difference in femoral head height (FHH) (p = 0.046), the change in femoral neck shaft angle (p = 0.003), the change in medial cephalic nail length (p = 0.033), and the change in tip-apex distance (TAD) (p = 0.002) were statistically significant compared to the relevant markers of imaging stability. Fracture stability had a substantial influence on Harris ratings at 3, 6, and 12 months following surgery, according to repeated measures analysis of variance (F(1,126) = 32.604, p < 0.001). The effect of time on the Harris score was similarly significant (F(1.893,238.508) = 202.771, p < 0.001). The observer intra-observer inter-group correlation coefficient (ICC) value was 0.941 > 0.75, the inter-observer ICC value was 0.921 > 0.75, and the intra-observer and inter-observer reliability were both good. CONCLUSION: The six-part classification of femoral intertrochanteric fractures based on 3D-CT images has broader guiding relevance for femoral intertrochanteric fracture stability analysis. Clinicians will find this classification simpler and more consistent than the AO classification.