Further researches showed that CDK5–ERK5–AP-1 axis could promote progression of CRC carcinogenesis and had a significant correlation in human CRC samples. In summary, this study revealed the functional and mechanistic links between CDK5 and the oncogenic ERK5–AP-1 signaling pathway in the pathogenesis of CRC. These findings suggest that CDK5 has an important role in CRC development and may serve as a potential therapeutic target for CRC.Abnormal expression of cyclin-dependent kinase 5 (CDK5) has been found in several human cancers, whereas the role of CDK5 in the malignant development of colorectal cancer (CRC) has not been well characterized. Here we investigated the role of CDK5 in CRC and found that its expression was much higher in CRC tissues than that in normal tissues with a higher expression level of CDK5 closely correlating to advanced American Joint Committee on Cancer (AJCC) stage, poor differentiation, increased tumor size and poor prognosis of CRC. Biological function experiments showed that CDK5 regulated CRC cell proliferation and metastasis ability. Whole-genome microarray analysis, co-immunoprecipitation,  Thus research of the molecular mechanism that is responsible for the initiation and progression of CRC about the biomarkers could help to identify potential biomarkers, which may facilitate efficient predictive and therapeutic strategies.Colorectal cancer (CRC) is one of the most common malignancies in the world involving progressive disruption of epithelial cell proliferation, apoptosis, differentiation and survival mechanisms.5 In the central nervous system, CDK5 has been proved as a key regulator of neuronal migration, synaptic activity and neuronal cell survival and death.8 Over the past decade, an increasing body of evidence has suggested that CDK5 may also have a significant role in the tumorigenesis of multiple organs, such as breast cancer, pancreatic cancer and neuroendocrine thyroid carcinoma.11 However, the knowledge on the role and underlying mechanism of CDK5 in CRC remains poorly unknown.Among the cyclin-dependent kinase (CDK) family, CDK5 is an unusual member with specific functions. Though CDK5 is ubiquitously expressed, previous studies about CDK5 were mainly focused on neuronal origin. Unlike other mitotic CDKs, CDK5 is activated by binding to p35 or p39.In the present study, we sought to investigate the clinicopathological significance of CDK5 in CRC and its role in CRC development. We found that CDK5 and its activator p35 showed higher expression levels in CRC tissues than paired normal tissues. In addition, high expression level of CDK5 was correlated to the aggressive characteristics (American Joint Committee on Cancer (AJCC), tumor differentiation, tumor size and nodal metastasis) and poor survival of patients. Furthermore, CDK5 might promote proliferation, tumor formation and invasion of CRC partly via modulating the ERK5–AP-1 signaling axis.14 Interestingly, CDK5 expression and its kinase activity was relatively higher in aggressive cell lines HCT116 and SW480 than that in less aggressive cell lines Caco-2 and Lovo (n=465) revealed a relative higher CDK5 and p35 mRNA expression in CRC tissue compared with normal tissue , tumor differentiation (P=0.029), tumor size (P=0.001) and nodal metastasis (P=0.027)   containing a total of 89 colon cancer patients using IHC staining . As a reP=0.027) . NotablyP=0.001) .P<0.001), tumor differentiation , tumor size , nodal metastasis  and CDK5 expression . Based on the results of the univariate survival analysis, multivariate survival analysis was performed. After adjustment, tumor size, tumor differentiation, AJCC stage and CDK5 expression were identified as covariates. Nodal metastasis was excluded from the multivariated survival analysis owing to its interactions with AJCC stage. The outcome suggested that AJCC stage  and CDK5 expression  were both considered as the independent risk predictors for poor overall survival : 3.790, 95% confidence interval (95% CI): 1.889–7.605; survival .To investigate the biological function of CDK5 in CRC cells, first we established stable CDK5 silencing cell lines using a specific shRNA in SW480, HCT116 cell lines and CDK5 overexpressing CRC cell lines with Lovo cell line . StrikinConversely, overexpression of CDK5 promoted an aggressive phenotype of the Lovo cell line by enhancing its cell proliferation ability, colony-formation density and soft agar colonies. .We also investigate the effects of CDK5 deregulation on cell cyle progression using flow cytometric analysis. The results demonstrated that depletion of CDK5 in CRC cell lines arrested HCT116 and SW480 cells at the G1/S-phase transition, whereas overexpression of CDK5 in Lovo cells accelerated the G1- to S-phase transition .in vivo metastasis assays. As shown in We then evaluated the effect of CDK5 on the metastasis of CRC cell lines. Results from transwell assays revealed that knocking down of CDK5 decreased migration and invasion ability of CRC cells, while overexpression of CDK5 achieved the opposite effect . Furtherz-score: −3.74, To further understand the underlying molecular mechanisms of CDK5 mediating carcinogenesis in CRC, we performed whole-genome expression microarray in HCT116/Scramble and HCT116/ShCDK5 cells. According to the analysis of Ingenuity Pathway Analysis (IPA) database, knocking down CDK5 remarkably downregulated a series of genes that involved in celluar proliferation, survival and movement . Besidesin vitro kinase assay showed that CDK5 directly phosphorylate ERK5 at Thr732 but not the canonical site of ERK5 at TEY microdomain , c-jun (r=0.732 P=0.025) and c-fos (r=0.867 P=0.002). These data further supported the notion that CDK5 could activate the ERK5–AP-1 signaling axis and lead to a poor clinical outcome in CRC.We further investigated whether CDK5 expression and ERK5–AP-1 activation are clinically relevant in human CRC. As shown in 15 this is the first study to investigate the clinicopathological significance of CDK5 in CRC. Our research has showed that high expression of CDK5 protein is significantly correlated with the aggressive characteristics  as well as poor survival of patients. Besides, a CRC microarray cohort from TCGA also verified our results. Similar to our results, in cancer such as lung cancer and pancreatic cancer, CDK5 and p35 overexpression were frequently but not solely associated with gene mutation.16 These researches indicated that CDK5 may serve as a universal tumor biomarker to identify patients with poor clinical outcomes.Although CDK5 protein level was previously observed in CRC,5 Recently, CDK5 has been proposed to possess other functions than that in nervous system, especially in cancer progression. Interestingly, CDK5 may have different roles during malignant development. Majority of studies supported that CDK5 might have a role in promotion of multistep process of tumor formation and progression, including transformation, proliferation, angiogenesis, invasion and metastasis.18 On the contrary, Cao et al.19 found that CDK5 was decreased in gastric cancer and overexpression of CDK5 could inhibit gastric tumorigenesis. Our research suggested that different levels of CDK5 and its kinase activity were detected in CRC cells and knocking down CDK5 would be associated with lower proliferation, metastasis ablility and arrest G1–S transition of CRC cells, while overexpression of CDK5 performed the opposite effect. Thus these observations suggested that CDK5 might function as a tumor promotor in human CRC.CDK5 was discovered in the early 1990 as proline-directed serine/threonine kinase in the CNS and emerged as a crucial regulator of neuronal development, function and disease.To further determine the mechanism as to how CDK5 promoted CRC cell proliferation and metastasis, we performed whole-genome expression microarray in CDK5 knocked down CRC cells. As expected, CDK5 controlled a cluster of genes and signaling pathways related to cancer proliferation and metastasis. Based on the analysis of IPA database, ERK5 signaling pathway ranked the top as the most highly inhibited and two of its downstream effectors, FOS and JUN, which comprised a key transcription factor activator protein 1 (AP-1), controlled a cluster of key oncogenic gene's expression. These data suggested a connective link between CDK5 and ERK5–AP-1 signaling axis. It is worth mentioning that data from genomic microarray showed that downregulation of CDK5 caused a series of gene alteration that belong to the downstream of ERK5 but not ERK5 itself, which mean that CDK5 might modulate ERK5 in a structurally modified manner at the protein level but not solely at the genetic level.20 During the past decade, the function of ERK5 signaling in cancer progression has been a hot research area. For example, inactivation of MEK5/ERK5 signaling pathway may reduce the expression of c-myc in CRC cells and finally supress cell proliferation, colony formation and cell invasion of CRC.21 A recent research also suggests that ERK5 signaling rescues intestinal epithelial turnover and tumor cell proliferation upon ERK1/2 abrogation, while a combination of MEK1/2 and ERK5 inhibitors show a more remarkable effect in inhibiting CRC cell growth.22ERK5 is an effector kinase of the canonical three-tiered MAPK signaling cascade comprising MEKK2/3, MEK5 and ERK5 itself.24 AP-1 possesses a specific phenotype that consists of either a Jun–Fos heterodimer or a Jun–Jun homodimer.25 AP-1 has been demonstrated to have a critical role in tumor promotion via modulating the genetic transcription of some important oncogenes, such as VEGFA, MMP1, c-myc and so on.28 Previous studies revealed that AP-1 could promote the proliferation ability and enhance tumorigenic properties of CRC cells.30 These researches suggest that the ERK5-AP-1 axis is a key signaling pathway in CRC progression, which is still needed more attention.Interestingly, among the downstream molecular effectors of ERK5 signaling, phosphorylation of ERK5 in its TEY microdomain area or c-terminal region with multiple sites, such as Ser706 and Thr732, is able to activate transcription not only by direct phosphorylation of transcription factors but also by acting itself as a transcriptional coactivator for one of its key downstream effector, the AP-1 transcription factor.in vitro and in vivo levels showed that CDK5 could modulate the ERK5–AP-1 axis. Although CDK5 has been found to closely link to some other MAPK signaling cascades in neurons,32 it is the first time to illustrate the relationship between CDK5 and ERK5 pathway. We found that CDK5 could directly phosphorylate ERK5 at Thr732, which suited the conservative sequence of CDK5(X-S/T-P-X).33 Further studies showed that knockdown of CDK5 decreased the phosphorylating level of ERK5 at Thr732, AP-1 and some of its target genes' expression, which was reversed when CDK5 was overexpressed without a ERK5 inhibitor in CRC cells. As a result, when CDK5 activated the ERK5–AP-1 axis, some important oncogenic genes, such as VEGFA, MMP1 and c-myc, were deregulating, which finally resulted in the malignant development of human CRC both in in vitro and in vivo experiments. On the other hand, the CDK5–ERK5–AP-1 axis showed a significant clinical relevance in human CRC. Thus it is clear that CDK5 may promote cell proliferation and metastasis partly by modulating the ERK5–AP-1 axis.As expected, our data from both the In conclusion, our findings help provide evidence that elevated CDK5 in human CRC has an important role in the acquisition of the progression as well as poor prognosis of the tumor. Furthermore, we also identified the CDK5–ERK5–AP-1 axis as a potential oncogenic pathway in CRC. This study not only further elucidates the precise role of CDK5 in the pathogenesis of CRC but also provides new insight into the biological basis of cancer progression and may help to contribute to more effective therapeutic strategies for CRC.Human CRC cell lines  were originally purchased from American Type Culture Collection . The cells were cultured in Dulbecco's modified Eagle's medium  supplemented with 10% FBS  and 1% penicillin/streptomycin (Invitrogen).Human CRC tissues and corresponding normal colon tissues for immunoblotting (freshly frozen) were provided by patients with CRC who had undergone surgical rsection in the Department of General Surgery, Nanfang Hospital, First Affiliated Hospital of Southern Medical University, Guangzhou, China. All patients provided written informed consent.The CDK5 shRNA sequence was sense 5′-TATAAGCCCTATCCGATGT-3′ and the control shRNA sequence was sense 5′-TTCTCCGAACGTGTCACGT-3′. The human CDK5 construct was generated by cloning PCR-amplified full-length human CDK5 cDNA with the transcript NM_004935. Both the shRNA sequence and the CDK5 construct were subcloned into lentiviral expression vector by GeneChem . The shRNA lentivirus and the control lentrvirus were transfected into the relative high expressing CDK5 cell lines HCT116 and SW480. The CDK5 low expressing cell lines Lovo was transfected with lentiviruses expressing CDK5 cDNA.34 The section of TMA was baked at 65 °C for 30 min. Then the section were deparaffinized with xylenes and rehydrated. After treatment with 3% hydrogen peroxide in methanol to quench the endogenous peroxidase activity, the section were submerged into citrate buffer and high-pressure boiled for antigenic retrieval, followed by incubation with 1% bovine serum albumin to block the nonspecific binding. Rabbit anti-CDK5  or rabbit anti-p35  was incubated with the section overnight at 4 °C. For negative controls, the rabbit anti-CDK5 or anti-p35 antibody was replaced with normal goat serum. After washing, the TMA section were treated with anti-rabbit secondary antibody . The tissue section were incubated with 3,3-diaminobenzidin and counterstained with hematoxylin, dehydrated and mounted. The section were reviewed and scored independently by two observers, based on both the proportion of positively stained tumor cells and the intensity of staining. The proportion of positive tumor cells was scored as follows: 0 (no positive tumor cells), 1 (<10% positive tumor cells), 2 (10–50% positive tumor cells), and 3 (>50% positive tumor cells). The intensity of staining was graded according to the following criteria: 0 (no staining); 1 (weak staining=light yellow), 2 (moderate staining=yellow brown), and 3 (strong staining=brown). The staining index was calculated as staining intensity score × proportion of positive tumor cells. Using this method of assessment, the expression of CDK5 was scored as 0, 1, 2, 3, 4, 6 and 9. Cutoff values for CDK5 were chosen on the basis of a measure of heterogeneity by log-rank test statistical analysis with regard to overall survival. An optimal cutoff value was identified: the score of ⩾4 was used to define tumors as high CDK5 expression and ⩽3 as low expression of CDK5.The TMA containing a total of 89 colon cancer patients, together with the data of pathological staging in accordance with TNM classification of the AJCC 2010 and overal survival time for all cases , was obt35 using anti-CDK5, anti-FAK, anti-p-FAK (Ser732), anti-PAK1, anti-p-PAK1 (Thr212), anti-ERK5, anti-p-ERK5 (Thr218/Tyr220) (Abcam), anti-p-ERK5 (Thr732), anti-p35 , anti-c-fos, anti-c-jun , anti-c-myc, anti-VEGFA and anti-MMP1 antibodies . Loading control was used with a mouse anti-β-Actin monoclonal antibody (Proteintech).Western blotting was performed as previously described,3/well). At each time point, cells were detected by the Cell Counting Kit-8 Kit following the kit assay protocol. Besides, the sensitivity of HCT116 and SW480 to CDK5-specific inhibitor ROS was also determined by CCK8 assay at various concentrations of ROS. The same concentrations of DMSO were used as control.The stable cell lines were seeded on 96-well plates at initial density of  and incubated at 37 °C in a humidified 5% COFour hundred cells were suspended in 2 ml complete medium containing 0.35% agar . Then the agar–cell mixture was plated on top of a bottom layer with 0.7% complete medium agar mixture. Twelve days later, the colonies were measured with an ocular micrometer ; colonies > 0.1 mm in diameter were counted. The experiment was repeated independently three times for each cell line.5 cells/well and cultured for 2 days. Cells were then collected and fixed with cold 70% ethanol for 24 h at 4 °C. Next, cells were incubated with 0.5 mg/ml of propidium iodide along with 0.1 mg/ml of RNase A. Cell cycle analysis was performed using flow cytometry .The effect of CDK5 on cell cycle progression was determined by flow cytometry using a Cell Cycle Detection Kit . The transfected cells were seeded in six-well plates at 2 × 10μm membranes were used . For invasion assays, The membrane was covered with 40 μl of the BD Matrigel (diluted 1 : 8 with serum-free medium) in advance. The stable cells (1–1.5 × 105) were seeded into the transwell chambers with serum-free medium while the lower chamber was covered with 500 μl medium supplemented with 20% fetal calf serum. After 48 h incubation at 37 °C, cells were harvested and fixed in 4% paraformaldehyde for 20 min and then stained with 0.5% crystal violet diluted in methanol for 25 min. The membrane was removed and mounted onto glass slides and counted (five × 200 random fields per membrane). Three independent experiments were performed and the data are presented as mean±S.D.For migration assays, the transwell chambers equipped with 8-6 cells were lysed using ice-cold RIPA buffer containing protease and phosphatase inhibitors. After a 30-min incubation at 4 °C, total extracts were clarified by centrifugation at 12 000 r.p.m. for 30 min. Then the cell extracts were precleared with 50 μl protein A+G agarose  for an hour at 4 °C, and the cleared extracts were immunoprecipitated with 2 μg of the indicated antibodies  and 50 μl protein G-agarose overnight at 4 °C. The immune complexes were washed three times with PBS buffer for 3 min each time. After the third wash, immunoprecipitates were re-suspended in SDS-PAGE sample buffer containing loading dye and used for western blotting as previously described.35Co-immunoprecipitations of endogenously expressed proteins were performed in HCT116 cells. In all, 5 × 10T732A (Thr732 to Ala), ERK5AEF . In addition, a construct containing wild-type ERK5 sequence was generated. All the versions were tagged with an HA epitope into a pBOBI lentivirus vector.Mutatgenesis of ERK5 was generated by Genesent . Briefly, the indicated amino acids were changed as follows: ERK5in vitro kinase assay, human 293 cell lines transfected with above lentivirus vectors were treated with BIX02189 under the indicated conditions, and then the cells were lysed using ice-cold RIPA buffer containing protease and phosphatase inhibitors and immunoprecipitated with anti-HA Mab-sepharose (7seaBiotech) and anti-HA antibody (Abcam) overnight at 4 °C. After washing three times with PBS, the eluted immunoprecipitates were incubated with 10 ng recombinant active CDK5/P35 protein or MEK5 protein  in kinase buffer (25 mMTris (pH 7.5), 2 mM DTT, 5 mM β-glycerolphosphate, 1 mM Na3VO4 and 10 mM MgCl2) along with 5 μM ATP for 30 min at 37 °C. Reactions were terminated by the addition of 20 μl 5 × loading buffer, and the samples were then resolved by immunoblotting. Phosphorylation of Thr732 and Thr218/Tyr220 of ERK5 was detected by corresponding specific phospho-antibodies.For the CRC cells were seeded in 12-well plates for 48 h. Next the cells were fixed in 4% paraformaldehyde and permeabilized in 0.5% Triton at 37 °C for 30 min. After blocking for 1 h with 5% BSA, the cells were incubated overnight with anti-p-ERK5 antibody . Cell incubated with anti-p-ERK5 antibody were then incubated at 37 °C with FITC-conjugated anti-rabbit IgG (Zhongshan Biotech). Then the cells were stained with DAPI. Images were captured under a fluorescence microscope. The experiments were performed at least three times.36 Biotin-labeled and unlabeled probes as competitor containing AP-1-specific binding were designed as the following sequence :5′-CGCTTGATGACTCAGCCGATCTGACTCA-3′ (forward); and 5′-TGAGTCAGATCGGCTGAGTCATCAAGCG-3′ (reverse).EMSA was performed as previous study using the LightShift Chemiluminescent EMSA Kit .4) were seeded in triplicates in 48-well plates and allowed to settle for 24 h. In all, 0.3 μg of REPOTM-AP-1-luc plasmid, or the control-luciferase plasmid, plus 30 ng of pGMR TK renilla plasmid  were transfected into CRC cells using the Lipofectamine 3000 reagent (Invitrogen). Forty-eight hours after transfection, luciferase and renilla activities were measured using the Dual Luciferase Reporter Assay Kit .Cells  and more details were shown in GSE81085. To assess the molecular signaling pathway change after knockdown of CDK5, data analysis, including canonical pathway and gene network interaction, was performed by the IPA. The P-value was calculated via the Ingenuity Pathways Knowledge Base-depedent Fisher's exact test.The microarray experiments were performed at Shanghai Biotechnology Corporation, Shanghai, China. Total RNA was isolated using the RNase Kit  from three replicate samples of the stable cell lines HCT116/Scramble and HCT116/ShCDK5. Agilent Whole Human Genome Oligo Microarray(4 × 44K)  was used for transcriptome analysis as previously described.in vivo, 2 × 106 HCT116/Sramble, HCT116/ShCDK5, Lovo/Vector and Lovo /CDK5 cells were injected into the tail vein of 4–6-week-old Balb/C athymic nude mice . All mice were housed and maintained under specific pathogen-free conditions. Two months later, the mice were killed and the lungs of them were dissected for futher analysis.To evaluate the metastatic potential to lung of cancer cells 6), including Lovo/Vector and Lovo /CDK5, HCT116/Sramble and HCT116/ShCDK5, on the right hind limbs of 4–6-week-old Balb/C athymic nude mice . All mice were housed and maintained under specific pathogen-free conditions. When tumors grew in one group injected with Lovo/CDK5 cells, BIXO2189 were used to inject at the positions of tumors ). The tumor volume was calculated using the following formula: tumor volume=4π/3 × (width/2)2 × (length/2), where the width and length were the shortest and longest diameters, respectively. After 3–4 weeks, the mice were killed during 21–30 days and tumors were then dissected and used for IHC staining as mentioned above (concentrations of different antibodies used for IHC staining are: anti-pERK5(Thr732):1 : 200; anti-c-fos:1 : 200; anti-c-jun:1 : 100; anti-VEGFA:1 : 150; anti-MMP1:1 : 250; and anti-c-myc:1 : 100). All animal experiments were approved by the Use Committee for Animal Care and preformed in accordance with the institutional guidelines.Xenograft tumors were generated by subcutaneous injection of CRC cells . Pearson's chi-square was used to analyze the relationship between CDK5 expression and the clinicopathological features of CRC. Survival curves were plotted by the Kaplan–Meier method and compared using log-rank test. The Cox proportional hazard model was used to calculate relative risk ratios. Univariate and multivariate survival distributions were compared using log-rank test. Two-tailed independent Student's


The iron-sulfur (2Fe-2S) binding motif CDGSH appears in many important plant and animal proteins that regulate iron and reactive oxygen metabolism. In human it is found in CISD1-3 proteins involved in diabetes, obesity, cancer, aging, cardiovascular disease and neurodegeneration. Despite the important biological role of the CDGSH domain, its origin, evolution and diversification, are largely unknown. Here, we report that: (1) the CDGSH domain appeared early in evolution, perhaps linked to the heavy use of iron-sulfur driven metabolism by early organisms; (2) a CISD3-like protein with two CDGSH domains on the same polypeptide appears to represent the ancient archetype of CDGSH proteins; (3) the origin of the human CISD3 protein is linked to the mitochondrial endosymbiotic event; (4) the CISD1/2 type proteins that contain only one CDGSH domain, but function as homodimers, originated after the divergence of bacteria and archaea/eukaryotes from their common ancestor; and (5) the human CISD1 and CISD2 proteins diverged about 650–720 million years ago, and CISD3 and CISD1/2 share their descent from an ancestral CISD about 1–1.1 billion years ago. Our findings reveal that the CDGSH domain is ancient in its origin and shed light on the complex evolutionary path of modern CDGSH proteins. This domain is characterized by the following consensus sequence, [C-X-C-X2-(S/T)-X3-P-X-C-D-G-(S/A/T)-H], in which the CDGSH sequence is underlined, and the 3Cys-1His 2Fe-2S coordinating amino acids (aa) are indicated in bold. It was initially annotated as a zinc finger binding domain, but was later shown to bind a 2Fe-2S iron-sulfur cluster7. CDGSH proteins can be classified into Class I CDGSH proteins that contain only one copy of the Fe-S binding domain, and Class II CDGSH proteins that contain two copies of the Fe-S domain1. In human, 3 different genes encode CDGSH proteins: CISD1 encodes mitoNEET (mNT), a homodimer that is anchored to the outer mitochondrial membrane (OMM) and is involved in diabetes, obesity, cancer, cardiovascular disease and neurodegeneration20. CISD2 encodes NAF-1, also a membrane-anchored homodimer that is localized to the ER, OMM and the membranes that connect them, and is involved in cancer, neurodegeneration, skeletal muscle maintenance, aging and the regulation of autophagy and apoptosis33. A NAF-1 dysfunctional variant was also found to be the causative agent of the human monogenic genetic disease Wolfram Syndrome 2 (WFS2) that is associated with juvenile diabetes, hearing deficiencies, neurodegeneration, blindness, and lower life expectancy38. NAF-1 and mNT were also shown to regulate mitochondrial iron and reactive oxygen species (ROS) metabolism, a function that was proposed to be conserved among plant and animal NEET proteins39. Both mNT and NAF-1 belong to the Class I family of CDGSH proteins1. In contrast, CISD3, the third CDGSH human protein, is different from CISD1 and 2 because it is a monomer that contains 2 iron-sulfur (2Fe-2S) clusters, and is hence a Class II CDGSH protein. CISD3 is not membrane anchored, and is localized to the matrix space of the mitochondria5. Very little is known about the function of CISD3 , but its expression level was found to be associated with tumorigenesis (http://www.proteinatlas.org). Furthermore, among the 3 human CDGSH proteins, it is the only one to be proposed as an essential protein40 (http://tubic.tju.edu.cn/deg/).The CDGSH domain is part of an iron-sulfur (2Fe-2S) binding motif that appears in several important human proteins e.g., NEET proteinsCyt-b5, thioredoxin, Fer4_19, Rieske and the Ferritin-like domain, indicating that it could be involved in various metabolic reactions in different organisms1. Perhaps the most important feature of this 3Cys-1His, 2Fe-2S-binding domain, demonstrated for the human CISD1 and CISD2 NEET proteins, is that it is both a relatively stable iron-sulfur binding domain, but at the same time it can participate in different reactions that transfer electrons and/or its entire iron-sulfur cluster to different electron and/or cluster acceptor proteins, respectively51. This feature may explain why the CDGSH domain is highly conserved from bacteria to human. In addition, it could serve as the basis for the participation of the CDGSH domain in many different important biological functions, as part of essential proteins. A recent study demonstrated, for example, that if the CDGSH domain of NAF-1 is mutated from a 3Cis-1His coordinating structure to a 4Cis coordinating one (a single aa mutation that stabilized the cluster 25-fold over), NAF-1 loses its key function in promoting cellular proliferation in cancer cells52. The important function of the CDGSH domain in human disease has also led to different attempts to target this domain with different drugs54.The CDGSH domain appears in multiple proteins that belong to bacteria, archaea and many different unicellular and multicellular eukaryotic organisms, often in combination with other important domains such as 1. Our study suggested that the Dictyostelium discoideum’s CDGSH proteins might be the closest to the ancient archetype of eukaryotic NEET proteins. We further suggested that mNT and NAF-1 emerged via gene duplication around the origin of vertebrates1. However, the evolutionary timings of these events were not determined. Furthermore, an in depth phylogenetic analysis of the CDGSH domain in bacteria and archaea was not performed.We recently used the three members of the human NEET protein family (CISD1–3) as guides to conduct a phylogenetic analysis of eukaryotic NEET proteins58, and that its early appearance in archaea and bacteria is associated with the ancient 4Fe-4S binding domain Fer4_19. We further show that a CISD3-like protein with two CDGSH domains on the same polypeptide (Class II), most likely represents the ancient archetype of CDGSH proteins. We also report that the human Class II (CISD3) protein is more closely related to the bacterial CDGSH protein (of proteobacteria) than to the archaeal Class II CDGSH proteins, and that the human Class I (CISD1/2) protein is more closely related to archaeal than bacterial CDGSH proteins. Using a molecular clock analysis we also show that the separation of the Class I and Class II eukaryotic CDGSH proteins could be traced to the origins of eukaryotic organisms, and that the human mNT and NAF-1 proteins diverged from their common ancestor ~650–720 million years ago (MYA).Here we address the two ends of the CDGSH evolutionary path: The origination of the CDGSH domain in prokaryotes (archaea and bacteria) and eukaryotes; and its divergence times, particularly during the appearance of vertebrates. We show that the CDGSH domain appeared early in evolution, probably linked to the heavy use of Fe-S driven reactions by early organisms1, representatives of both Class I and Class II CDGSH proteins could be found in Archaea  4Fe-4S cluster binding domain57. As in eukaryotes1, several classes of Archaea lack CDGSH proteins suggesting that some environmental adaptations or metabolic dependencies may not require CDGSH proteins. To conduct a more detailed analysis of CDGSH proteins in archaea, we constructed a phylogenetic tree using PhyML (see Methods) for several different representatives of archaeal CDGSH proteins using the human CISD3 as an outlier to root the tree , whereas the Class I CDGSH proteins of Thermoprotei appeared to be the among the least derived (the term “derived” refers to branching events in a lineage following divergence from the last common ancestor in a phylogenetic tree). We also retrieved a time tree from TimeTree.org for these proteins . This finding could suggest that Class II with the Fer4_19 and Class I CDGSH domain structure proteins were among the earliest to appear in Archaea. It is also possible that the Thermoprotei lineage initially had a Class II with the Fer4_19 domain protein which was lost in the course of evolution, and only the single domain CDGSH protein was retained. Interestingly, very few examples of Class II CDGSH proteins without a Fer4_19 domain could be found in archaea. The main forms of archaea CDGSH proteins included therefore the Class I and the Class II CDGSH proteins that contained the Fer4_19 domain .The CDGSH domain is found in the genomes of extant archaea belonging to different taxa Fig. . SimilarDeinococcus-Thermus. In many of the bacterial phyla the CDGSH domain appeared in association with other domains such as Fer4_19, Glu_synthase, Ferritinlike, and Rieske_2, suggesting that it is associated with many different pathways that are mostly linked to iron and iron-sulfur metabolism  domain  was more similar to the CDGSH domain that is closer to the N-terminal of the Class II proteins , than to the CDGSH domain that is closer to the C-terminal of Class II CDGSH proteins , we generated two rooted phylogenetic time trees that were very similar in their topology  domain , suggesting that some aspects of CDGSH function could be different between prokaryotes and eukaryotes.Our phylogenetic analysis of the CDGSH domain in prokaryotes revealed that it is highly conserved and widespread among many phyla of bacteria and archaea, suggesting that it evolved early during the emergence of life on Earth Figs –S8. Its ain Figs –S4 demonaea Figs -S4, we c24 and on the human and bacterial CISD3 proteins68, our previous phylogenetic analysis of eukaryotic CISD proteins1, and our current analysis of these proteins in archaea, bacteria and eukaryotic organisms, reveal an interesting property of CDGSH proteins. When appearing as a Class I single domain CDGSH proteins such as mNT and NAF-1, CDGSH proteins function as homodimers. In contrast, when appearing as a Class II CDGSH proteins that have two CDGSH sequences on the same polypeptide, CDGSH proteins function as a monomer. Furthermore, CDGSH proteins with 3 or more CDGSH domains on the same polypeptide were not found in our current or previous analysis of CDGSH proteins in genomes from different life domains1. It is therefore possible that CDGSH proteins require two CDGSH 2Fe-2S clusters in close proximity to each other to be able to function in different biological systems. Although this hypothesis, which is based on structural and phylogenetic studies, is highly speculative and would require additional structural and evolutionary studies to be validated, it nevertheless bears importance when attempting to speculate on the origins and functions of ancient CDGSH proteins. Did these proteins originate as a Class I single domain, or did they originate as a Class II double domain? Although we may never know the answer to this question, our findings that bacteria primarily contain Class II CDGSH domain proteins, and that the ancient archetype of CDGSH proteins in bacteria could potentially be a Class II protein associated with a Fer4_19 domain  was embedded within an archaeal clade, with Halobaculum gomorrense  as the nearest neighbor  to a bacterium (Asticcacaulis benevestitus). Furthermore, as both these strains are aquatic isolates and dwell in hypersaline environment, their shared ecology might have facilitated gene exchange including of those harboring CDGSH domains70.Our findings that proteobacterial Class II CDGSH proteins are more similar to human and slime mold CISD3 proteins than to archaeal CDGSH proteins Fig. –S6, sugg24 might have independently occurred after bacterial and archaeal/eukaryotic lineages diverged from their common ancestor, and is currently found primarily in archaea and eukaryotes. Because many of the Class II CDGSH proteins of both bacteria and archaea contain the Fer4_19 domain, but eukaryotic Class II CDGSH proteins do not, it is possible that archaea and bacteria Class II proteins are related. In contrast, all eukaryotic Class II CDGSH proteins could have evolved from an ancient proteobacteria Class II CDGSH protein that might have lost the Fer4_19 domain or more likely, this domain got lost after the primary endosymbiotic gene transfer event. It is also possible that once Class I CDGSH proteins evolved, some organisms, for example plants, or certain bacterial and archaeal lineages, lost the Class II domain protein and retained only the Class I CDGSH protein. The model described above suggest that the evolution of a two domain CDGSH protein via domain duplication preceded the evolution of the single domain CDGSH protein that requires a homodimeric structure to function. Because a simple domain duplication rather than the emergence of mechanisms for two identical proteins to dimerize (likely to require a stepwise evolutionary process involving changes to many different amino acids at the surface of the protein) appears more parsimonious and thus plausible. It is reasonable to speculate that the function of the Class II proteins was initially established through domain duplication. This event was then followed by the more complex process of Class I homodimer protein evolution (a single domain protein that could function as a homodimer). Once this new form of protein (Class I homodimer) was established, the Class II protein could have been lost in some lineages, as is likely the case in plants1. The high prevalence of proteins containing the CDGSH domain in bacteria and archaea is similar to that of proteins containing other important domains such as the catalase heme or the Fer4_19 domains, indicating that the CDGSH domain could have played an important role in evolution  for determining the divergence time of the CISD genes66. As previous analysis had reported Dictyostelium discoideum (a protist slime mold unicellular cell) as the possible representative of the most ancient CISD gene in eukaryotes1, we used the Dictyostelium CISD (XP_647247.1) sequence as the query sequence to search for potential bacterial CISD homologs in the non-redundant database using PSI-BLAST71. PSI-BLAST iterations were performed until no new BLAST hit was retrieved. All sequences thus obtained were subjected to domain analysis using PFAM, which utilizes profile hidden Markov model to predict the domain architecture of the protein sequences72. Sequences with at least one CDGSH domains were kept, and incomplete and partial sequences were discarded. A total of 494 bacterial sequences representing different lineages were selected for further analysis  to remove poorly aligned regions in order to obtain high quality alignments74.For the above three sets representing bacterial, archaeal, and eukaryotic CISD sequences, multiple sequence alignment was performed for each using command-line multiple alignment program MUSCLE with default options64. The time calibration points for each organism were obtained from the TimeTree website www.timetree.org59.Multiple sequence alignments of eukaryotic CISD sequences were analyzed using BEAST for the estimation of divergence times. Multiple combinations of population size change and molecular clock models were assessed in order to find the best-fit model. Among the models tested, the combination of a constant/exponential population size model and a relaxed uncorrelated log-normal clock with high estimated sample size (ESS) yielded the highest Bayes factor  calculated as the 95% highest posterior density (HPD) interval (upper-lower). The final MCC tree was visualized and edited with the program FigTree (http://tree.bio.ed.ac.uk/software/figtree/)75. The inferred time of divergence from an ancestral node is indicated next to each internal node in Fig. All BEAST Monte Carlo Markov Chain (MCMC) simulations were run for at least 50 million steps, with subsampling at every 1,000 steps. The trees generated by BEAST were summarized by a single maximum clade credibility (MCC) tree using TreeAnnotator76. For statistical reliability, the following tests were used: an approximate likelihood-ratio test (aLRT) based on logarithm of the ratio of likelihood computed for the current tree and that of the best alternative, and a Bayesian-like transformation of aLRT (aBayes). To estimate the optimal model of substitution, ProtTest was used for each alignment77. ProtTest indicated the WAG amino acid model with gamma distribution shape parameter (WAG + G) and the WAG amino acid model with invariable gamma distribution shape parameter (WAG + I + G) as the best fitting models among the 112 examined evolutionary models, based on Akaike information criterion (AIC) statistics, for archaea and bacteria respectively. The trees were visualized and designed with iTOL (Interactive Tree of Life) web-server78. The domain organization for each sequence was appended at the end of terminal branches, using iTOL, as shown in Supplementary Figs PhyML version 3.0 was utilized to generate maximum-likelihood trees for bacterial and archaeal CISD sequences59. However, the divergence times for multiple bacterial and archaeal organisms were not found in the TimeTree website. Nevertheless, we ensured that at least one organism from a class or phylum is represented in the time-tree.To generate an evolutionary timescale for the bacterial and archaeal organisms represented in our analysis, we generated time-trees using TimeTree.org website. The complete lists of representative bacterial and archaeal organisms Tables  and S2 wAll data used in this study is publically available. All data or tools generated by this study will be made available upon request.Supplementary Material


Covalent organic frameworks (COFs) are newly emerged crystalline porous polymers with well-defined skeletons and nanopores mainly consisted of light-weight elements  linked by dynamic covalent bonds. Compared with conventional materials, COFs possess some unique and attractive features, such as large surface area, pre-designable pore geometry, excellent crystallinity, inherent adaptability and high flexibility in structural and functional design, thus exhibiting great potential for various applications. Especially, their large surface area and tunable porosity and π conjugation with unique photoelectric properties will enable COFs to serve as a promising platform for drug delivery, bioimaging, biosensing and theranostic applications. In this review, we trace the evolution of COFs in terms of linkages and highlight the important issues on synthetic method, structural design, morphological control and functionalization. And then we summarize the recent advances of COFs in the biomedical and pharmaceutical sectors and conclude with a discussion of the challenges and opportunities of COFs for biomedical purposes. Although currently still at its infancy stage, COFs as an innovative source have paved a new way to meet future challenges in human healthcare and disease theranostic. However® . Organic® , while i® . Metal-o® . Thus, m−3 [Covalent organic frameworks (COFs) are new emerged crystalline porous polymers with periodic skeletons and large surface areas . Much si−3 .Since first reported in 2005 , COFs haIn this review, we cover the developments of COFs and summarize the applications of COFs in drug delivery, photothermal therapy, photodynamic therapy, biosensing, bioimaging and others , which are generally formed through reversible organic reactions. Different from traditional interactions, DCBs possess robustness of covalent bonds and error-checking capability of reversible bonds at the same time , which e2O was added, HHTP-DPB COF resolved 50% within 11 s and resolved 90% within 1 min [The first generation of COFs are based on boron acid-based building blocks and reported by Yaghi and co-workers . Boronicin 1 min . More thin 1 min ,94.To alleviate this drawback, Yaghi and co-workers designed borosilicate-linked COFs (COF-202) by condensation of boronic acids and silanols . B–O–Si 3 hybridized boron anionic centers [In 2016, spiroborate-linked ionic COFs (ICOFs) have been synthesized by incorporating cations to make boron atoms become sp centers . With thThe most representative C–N bond linked COFs are imine-based COFs , which were designed by Yaghi and co-workers . These CTo improve the chemical stability of imine-linked COFs, Banerjee and co-workers incorporated hydroxyl (–OH) into DhaTph COFs and created intramolecular O–H···N=C hydrogen bonds . The hydIn hexagonal imine-linked 2D COFs, each macrocycle contained 12 polarized C=N segments, which were composed of positively charged carbon and negatively charged nitrogen. Jiang and co-workers proposed that it was the electrostatic repulsion caused by these charged groups that destabilized the layered structure. Thus, they incorporated electron-donating methoxy groups into the pore walls of imine-linked COFs (named as TPB-DMTP-COFs) to reduce the electrostatic repulsion . After dp-phenylenediamine (Pa-1) and 2,5-dimethyl-p-phenylenediamine (Pa-2), respectively [Another method for reducing hydrolysis of imine-linked COFs is through irreversible enol-toketo tautomerism ,123,124.ectively . The whoectively . These cJiang and co-workers introduced conjugation effects into COFs to improve their hydrolytic resistance. Azine-linked COFs are connected by linear and conjugated linkages (–C=N–N=C–) through condensation of aldehyde and hydrazine and show excellent stability in water, acid (1 M HCl) and base (1 M NaOH) . PhenaziTriazine-linked COFs, also known as covalent triazine-based frameworks (CTFs), was first prepared by Thomas and co-workers through the cyclotrimerisation of nitrile building units . The ini2/g) but their preparation was simple and cheap.Zhu and co-workers designed melamine-based COFs by the polycondensation of terephthalaldehyde and melamine . These fp-bromo-benzene boronic acid [The first C–C bonds linked COFs were prepared by Ullmann coupling reaction under ultrahigh vacuum (UHV) . It can nic acid , 4,4″-dinic acid  and benznic acid , were us2 carbon-conjugated COFs (sp2c-COF) by C=C condensation reactions between aldehydes of tetrakis(4-formylphenyl)pyrene and acetonitriles of 1,4-phenylenediacetonitrile [2c-COF was long-term stored upon the air and stable in water, acid, base and various organic solvents.C–C bonds were also applied to the construction of conjugated COFs. Jiang and co-workers prepared sponitrile . The sp2Beside above-mentioned linkages, several other bonds were also used to construct COFs. Borazine-linked COFs are synthesized by thermal decomposition of arylamine-borane or -borontrihalide adducts, which possess high thermal stability (~420 °C) . AzodioxHetero linked COFs are prepared by two or more types of covalent bonds, such as imine and boronate ester bond , imine aNo matter for which kind of materials, a facile and productive method can be intriguing and makes it possible for industrialization production. Besides that, some special processes are also needed to build ideal structures. Since Yaghi and co-workers prepared the first COF under solvothermal conditions , many sy2-carbon [Solvothermal synthesis is the first and also the most widely used method to prepare COFs . It is s2-carbon  and bora2-carbon .A classical solvothermal approach is as follows. Designated monomers are dispersed in certain solvents, which are often mixed solvents. After several freeze-pump-thaw cycles, the system is sealed to preserve the produced water molecules to keep the reversibility of the reaction. After reacted under a certain temperature  for several days (from 2 to 8 days), the crude products will be precipitated from solution as solid powders. The designed COFs will be obtained after washed with organic solvents, e.g., acetone and dried under vacuum. o-dichlorobenzene [N-methyl-2-pyrrolidone/isoquinoline [n-butanol/o-dichlorobenzene [o-dichlorobenzene was the optimal combination for NiPc COF and 2:1  was the best ratio. In another study, they synthesized ZnP-COF in the mixture of mesitylene and dioxane with a different ratio [v/v). Scanning electron microscope (SEM) images showed that the resulted particles were in uniform cubic shape under this ratio. Besides, the reaction time and the dose of catalyst also have effects on the structure of COFs, too [In solvothermal synthesis, solvents play a crucial role in the formation of COFs, which should solve the monomers but not fully dissolve them . Differeobenzene , mesityluinoline , ethylenuinoline , n-butanobenzene  and so oobenzene . PXRD rent ratio . The cryOFs, too ,153.It is worth noting that, the solid concentrations were generally only 2% and a great deal of organic solvents were needed in the solvothermal synthesis process, which may cause environmental pollution . To solvSince the crystalline COFs are controlled by thermodynamic equilibrium, solvothermal synthesis can provide the system with enough energy to pass the barrier of Gibb’s free energy and form crystals more easily . But the2 at 400 °C and reacted for 40 h. In this process, ZnCl2 works as solvent as well as catalyst, so that the cyclotrimerisation reaction becomes reversible. When the system is cooled down to ambient temperature, the salts are washed out and the crystalline CTFs are prepared after purification.Ionothermal synthesis was used to prepare triazine-linked COFs and was first reported by Thomas and co-workers . NormallCompared to boronate-ester or imine linked COFs prepared by solvothermal synthesis, CTFs show poor crystallinity since the conditions of reversible reaction are very harsh. Besides that, only a few monomers are suitable for this method because the monomers should have good solubility in ionic melt and will not be decomposed under this high temperature . Thus, i2/g) is a bit higher than that prepared via solvothermal method (1590 m2/g) [Microwave synthesis has a long history in organic chemistry  but it w90 m2/g) , which i90 m2/g) . In addi90 m2/g) ,158, micl method 0 m2/g [190 m2/g) , triazin90 m2/g)  and mela90 m2/g) ,134 baseCompared to solvothermal synthesis, microwave synthesis uses less time and removes impurities more effectively, showing an attractive prospect.In general, building blocks are dissolved in solvents to prepare COFs, which can be limited by the physicochemical properties of monomers and the dissolving capacity of solvents. The residual organic solvents may also feature the products with toxicity . Mechanop-toluene sulfonic acid (PTSA) in sequence and mixed well by grinding. And then, a little water was added to make the mixture converted into dough. After heated at 170 °C for 1 min, the deep reddish powders were dipped into hot water to separate the designed COFs. These COFs featured highly crystalline, ultrahigh porosity and high surface area. What’s more, COFs will be molded into any desired shapes and sculptures through heating, mimicking the ancient terracotta process, as shown in In 2013, Banerjee and co-workers were the first to build β-ketoenamine-linked COFs by manual grinding in a mortar and pestle . It was 3) and bulking agent (ZnCl2) were mixed and milled in a planetary ball mill for 1 h, as shown in As mentioned above, CTFs were synthesized by ionothermal method in the beginning . Later, In brief, mechanochemical synthesis is a user-friendly, eco-friendly and time-saving alternative to produce COFs in a massive scale. It provides us a powerful method to realize the industrial production of COFs.m-cresol or DMSO as solvent instead of traditional mixture solvent and prepared RT-COF-1 in minines [3 was used to replace acetic acid, the frameworks were synthesized within 10 min and possessed a large specific surface area [Room-temperature synthesis was first mentioned in the review of Wang and co-workers , though  minines ,166. Lat minines . When Scace area .Room-temperature synthesis avoids strict reaction conditions but only –C=N– linked COFs have been prepared by this way. The generalization of this facile route still remains challenges.Interface synthesis is a bottom-up method to prepare COF films at the interface of solid-liquid, solid-vapor, liquid-liquid and liquid-air interface.Solid-liquid interface synthesis is often assisted with solvothermal method  method ,180,181.OFs, too ,188.Liquid-liquid and liquid-air interface synthesis are both used to prepare free-standing films that can be transferred onto other substrates ,191,192.3O4 [Apart from films, interface synthesis is also used to construct frameworks on the surface of other particles, such as Fe3O4 ,194 and 3O4 ,196.Interface synthesis is an ideal strategy to prepare well-structured COFs and great progress has been made in the past several years. However, the production capacity of this method is rather low.Beside above-mentioned methods, there are still some other ingenious approaches to build COFs.Shortly after COFs were first synthesized under solvothermal condition , LavigneBased on their former works ,124,164,Based on mechanochemical synthesis, Liu and co-workers synthesized nanofibrous COFs through a vapor-assisted solid-state approach . Brieflyv/v) or analogous solvents, were sealed and left undisturbed under room temperature. After 48 h, the yellow crystals were collected by filtration. This approach produced large single crystals with significant porosity under mild conditions.Normally, COFs are microcrystalline powders but Wuest and co-workers prepared a monocrystalline covalent organic network by reversible self-addition polymerization . The proCTFs also can be synthesized via lower temperature polycondensation approach . Based o3·OEt2 as a catalyst, deprotection and esterification were performed at the same time to obtain COFs. Bein and co-workers used boronate ester as starting materials to build BTD-COFs through the transesterification process [tert-butyloxycarbonyl (Boc) groups to protect amine building blocks to improve the crystallinity of COFs [Besides changing reaction conditions, yield and crystallinity also can be improved by changing precursors ,202,203. process . BTD-COFIn conclusion, enormous progress has been done in the synthesis of COFs during the past dozen years and researchers have never ceased efforts to explore more effective ways.COFs are characterized by high surface area and porous structure which make them ideal carriers for adsorbates ,58, cataTP-ANDI-COF/DTP-APyrDI-COF  [For designed COFs, the shapes and sizes of pores are determined by the geometrical configuration of building blocks . Jiang am, 2013) .Lavigne and co-workers adjusted the pore size by incorporating alkyl chains on the walls . They inPore volume is another important factor for loading capacity of carriers. As for a certain COFs, their pore sizes and structures are constant but their pore volumes can be changed by managing interlayer interactions ,227.In general, the synthesized COFs are irregularly shaped bulks, which limit their serviceable ranges especially in vivo application. However, COFs are hard to be reshaped, as they are insoluble in various solvents and also cannot be melted under high temperature . At one The most studied regular morphology of COFs is nanofilm. Normally, it can be divided into two strategies, namely bottom-up and up-bottom methods. Bottom-up method to prepare COFs at the interface of solid-liquid , solid-v2 [2O and DMF [Zamora and co-workers suspended COF-8 powders in dry dichloromethane and sonicated them for 15 min under the frequency of 24 kHz . After c2 . Interes2 . Dichtel and DMF . Banerje and DMF . What’s  and DMF .The low yield of COF films is still a major problem remained to be solved. Up-bottom method is easy to handle and suitable to most of COFs but the morphology of products is hard to control. Bottom-up method can make up for this defect and shows more promising perspective.The first class of regular bulk COFs was reported by Jiang and co-workers. In 2008, they built belt-shaped pyrene-based COFs, TP-COFs . And latLiu and co-workers put forward a dissolution-recrystallization growth mechanism based on the dynamic imine bonds, as shown in tert-butyloxycarbonyl (Boc) groups, COFs were synthesized in homogeneously. Based on this result, Yaghi and co-workers used Boc-protected amine to build crystalline woven COF-112 which was around 200 nm [3O4 can fabricate hybrid nanoparticles no more than 200 nm [COFs are also can be synthesized as uniform nanoparticles. When the amine building blocks were protected by d 200 nm . Besidesd 200 nm . They add 200 nm . Yan andd 200 nm . Nano-sin 200 nm ,194,195.n 200 nm .To summarize, some morphologies of COFs have been reported though a few of them has been unraveled their formation mechanism. It is important to uncover the secret of COF formation and design more desired nanostructures.Modifiability is one of the most fascinating characters of COFs, which can improve their stability, adjust their structural characters and feature COFs with some special functions. A recent study from Smaldone and co-workers concluded that even small structural differences in building blocks have a tremendous effect on the crystallinity, surface area, pore structure and luminescence properties of COFs . So thatSince COFs are consisted of building blocks, it is a straightforward way to functionalize COFs through changing their skeletons, which often makes COFs possess excellent photoelectric property. The first luminescent COFs was reported by Jiang and co-workers in 2008 . They inPorphyrin and their derivatives have proven to be intriguing functional building blocks for COF due to their unique photophysical properties, structural robustness, rich metal coordination chemistry and redox exchange behaviors . Yaghi aMuch similar to porphyrins, phthalocyanines are also square planar 18π aromatic macrocycles  and worktrans-to-cis photoisomerization under irradiation and can be recovered after heating [Azobenzene (AZO) is a widely studied photo-responsive unit, which can be performed  heating . A serie heating ,267,268. heating ,267,268. heating . Upon ph heating .2 was tunable with different counter cations.Efforts are also devoted in the increase of COFs’ conductivities. Incorporation of tetrathiafulvalene can provide COFs with tunable electrical conductivity ,270,271.2 and anionic pollutants [In addition, Banerjee and co-workers used guanidinium halide as building blocks to make COFs possess self-exfoliated and antibacterial property . Wang anllutants . Moreovellutants ,282.As we have mentioned earlier, incorporation of alkyl groups will tailor the pore size  and impr3-BDBA), which worked as blocks to build COFs with HHTP [60) were linked with phthalocyanine COFs to form donor-acceptor heterojunctions [l-tartaric anhydride ((+)-Ac-L-Ta) on COFs, which was used for chiral separation [One of the most studied methods is through azide-alkyne cycloaddition, which is known as a classical post-synthetic approach ,249. As ith HHTP . After tith HHTP . And eleunctions . Pyrroliunctions . Thus, Junctions ,248. Besparation . Zhao anparation . Cui andparation .Dichtel and co-workers developed a general method to functionalize 3D COFs, named as truncated mixed linker (TML) approach ,289. It A two-step post-synthetic method has been developed to obtain amide functionalized β-ketoenamine COFs . At firsN-hexylmaleimide molecules, which would disturb the π-π stacking interactions and result in the exfoliation of individual layers [2) with CONs. At last, folic acid was conjugated to CONs with the EDC/NHS coupling reaction, as shown in In addition, Banerjee and co-workers used cycloaddition reactions modified anthracene-containing COFs with l layers . By thisl layers . The firSmaldone and coworkers incorporated fluorine into pore walls and improved the crystallinity, surface areas and porosity of these COFs, compared to the non-fluorinated analogue ,296. TheApart from covalent bond, coordinate bond and physical interaction are also used to modify COFs by doping inorganic ions or particles.2 uptakes [2 and CH4 if counter-cations were lithium ions [The first metal-nanoparticle-hybrid COFs were reported by Smit and co-workers in 2009. They doped Li atoms into 3D COFs to improve the capacity of hydrogen adsorption and storage . Goddard uptakes . What’s ium ions .2 [2(acac)2 [2 to CO by electrocatalysis in water [Hybrid COFs are widely studied in catalysis. Wang and co-workers used the nitrogen atoms of imine-bonds to incorporate palladium acetate into COF-LZU1 and the resulting COFs showed excellent catalytic activity for the Suzuki-Miyaura coupling reaction . Similar2  and Zhan2(acac)2 . Yaghi, in water . Co(II) in water . Triazinin water . These ain water , which w2 production [More than a coordinate bond, Banerjee and co-workers directly immobilized Au and Pd nanoparticles into COFs by in situ reduction ,303. Thioduction . Similaroduction ,307,308.12O403− [Dopants can impact the conductivity of COFs. Sulfur was impregnated in COFs, e.g., CTF-1 , Azo-COF12O403− ,314,315.12O403−  and COF-12O403− . Jiang a12O403− .Along with those great progresses, COFs possess multiple prominent advantages that allow these materials for theranostic applications.(1) Dynamic covalent linkages for stimuli-responsibility and biodegradationInstead of traditional covalent bonds, coordinate bonds and physical interactions, COFs utilize dynamic covalent bonds as linkages, which can keep their structure in normal conditions and be broken by stimulations such as acid . These c(2) High surface area and large pore volume for high loading capacity2/g and COF-103 is 4210 m2/g; the pore volume of COF-103 is 1.66 m3/g and COF-10 is 1.44 m3/g [Due to their unique framework structures, COFs possess high surface areas and pore volume. For example, the BET surface area of COF-102 is 3472 m.44 m3/g ,207. The.44 m3/g ,32. What(3) Tunable pore structure for controlled releaseAs described in (4) Excellent photoelectric properties for biosensing and bioimagingMostly, COFs contain π-conjugated system and laminated structures, which feature them with excellent photoelectric properties. For instance, polycyclic aromatics units  can make COFs emit fluorescence and electron donors or acceptors units  can endow COFs with proton conductivity. Based on those properties, COFs show a great potential in biosensing and bioimaging.(5) Unique tailorable attributes for desired performanceSince COFs consist of building blocks with high tailorability, extensive materials will be designed as building blocks and introduced into their skeletons. By this way, COFs can exhibit various desired performance, such as produce singlet oxygen for photodynamic therapy . What’s (6) Outstanding modifiability for versatile functionalitiesModification is an effective way to enhance the applicability of COFs. Inspired by other nanoparticles, surface modification can improve their biocompatibility and targeting ability ,321. SinDue to their great potentials and unique features in diagnosis and treatment, COFs have been explored in drug delivery, photothermal therapy, photodynamic therapy, biosensing, bioimaging and other biomedical applications. Even though in their early stages, COFs have attracted intensive attention of researchers and a surge of studies have been reported in the past several years.The early example of applying COFs to drug delivery was reported by Yan and co-workers , who desPorphyrin-based covalent triazine frameworks (PCTFs) were also used in drug delivery . The autAlthough a number of studies were carried out to investigate in vitro drug release from various COFs, few works are focused on the in vivo biocompatibility and cytotoxicity. Zhao and co-workers conducted cell experiments with two imine-linked 2D COF, PI-2-COF and PI-3-COF . Both twStimuli-responsibility is one of most fascinating properties to achieve real-time monitoring and on-demand drug release ,326. BesBanerjee and co-workers did a series of exploration about imine based COF for drug loading and release ,116,198.Apart from carrying anti-cancer drugs, functional COFs can kill tumor cells by themselves. Bhaumik and co-workers reported an interesting work in which phloroglucinol contained COFs were used as anticancer agents . They apABBA was linked by boroxine bonds, neither loading efficacy nor release rate was studied. The pore sizes are most designed to fit the model drug but the morphologies of these carriers are various instead of uniform nanoparticles, which need further exploration. Generally, the drug loading efficacy (DLE) of COFs was rather high and exhibited pretty well biocompatibility. Although COFs have shown their pharmaceutical potential, several limitations markedly hamper their clinical translation. The intricate structural characteristics demand careful engineering of the COFs in order to realize the desirable effect. The complexity of the COFs preparations caused a key issue needed to be addressed in the scale-up production and batch-to-batch reproducibility. Challenges still exist in terms of delivery of the cargo to the targeted site as well as efficient clearance of the COFs once they have finished their mission in vivo. Although several studies have tested the efficacy of COFs formulations and their safety, few studies on their long-term accumulation and degradation profiles were carried out. However, we believe that continuous improvements in the design and detailed investigations on their in vivo behaviors will help to take COFs as nanocarriers from bench to bedside.Herein, we summarized those works in Besides chemotherapy, COFs also can be used for photothermal therapy (PTT)  and phot3O4 nanoclusters for PTT, as shown in 3O4@COF microspheres increased the system temperature by 25 °C  under photoirradiation and widely studied as photosensitizers for PDT [Porphyrin and its derivatives can generate singlet oxygen  . Since gCovalent organic frameworks, a representative material of porous organic polymers, have been widely explored in various fields and play an important role in nanotechnology. In this review, we present the significant developments of COFs formed by dynamic linkages, synthetic method, pore design, morphological control and functional modification. With these achievements, COFs exhibit numerous notable advantages, such as stimuli responsiveness, biodegradability, high surface area, large pore volume, tunable pore structure, photoelectric properties, unique tailored attributes and outstanding modifiability, which pave a way for this new porous framework to be used for biomedical purposes. Most importantly, the complementary functional design of skeletons and high flexibility in the control of pore geometry may provide a powerful means of exploring COFs for challenging biomedicine issues. Especially for the unique photoelectric property, COFs are expected to provide an ideal theranostic platform due to the convenience and ability to integrate the drug therapy and photoelectric diagnostic function in one COFs-based system. As a result, it was no wonder, although at its fancy stage, that the applications of COFs in drug delivery, photothermal therapy, photodynamic therapy, biosensing, bioimaging and other possible diagnosis have significantly increased and received increasing attention with the development of new COFs materials. However, despite their many favorable characteristics and great progress, most of the currently available COFs face many challenges in terms of preparation complexity, regulated morphology, hydrolytic stability, targeted delivery, controlled release, long-term biocompatibility, which indeed impair their practical applications in biomedical and pharmaceutical fields. As a result, considerable efforts are urgently required to design and fabricate COFs with well-defined morphologies and desirable properties. Especially, future work need to focus the following issues.(1) Scale-up preparation of COFs with controlled and uniform morphologyDespite the great progress made in the field of COFs design and preparations, none of the COFs and corresponding preparation methods are perfect. Firstly, as a class of crystalline and porous materials, the crystallinity and porosity of COFs are of central importance for many applications. Especially for pharmaceutical and biomedical applications, the uniformity in the materials properties and therapeutic performances and the reproducibility in the preparation process and product quality are two very important requirements. However, due to the complexity in the molecular structure of COFs and high heterogeneity in reaction environment, the biomedical applications of COFs not only face the almost inevitable crystal defects, stacking faults, pore dislocations and uncontrolled morphologies but also need to address the scalable production and batch-to-batch reproducibility. More importantly, through the existing approaches, it is still very hard to prepare stable COFs with a uniform size in the nano range. And thus, it is imperative to develop a simple and effective method to construct nanoscale COFs with precise crystal structure and well-defined pore geometry in a scalable process, achieving high uniformity, reproducibility and thus high-performance of the synthesized COFs. Secondly, the building blocks for constructing COFs are usually expensive or not commercially available. The most common starting materials are mainly a variety of polycyclic aromatic derivatives with multiple reactable groups, whose preparations generally involve multistep tedious reactions under drastic reaction conditions and complicated purification steps. Especially, these COFs materials and their degradation products always suffer from high toxicity, making them unsuitable for biomedical applications. The rational design and optimization of the building blocks in COFs have become a focus for research in the biomedical fields to enhance COF biosafety.In addition, it was worth pointing out that, at present, there are still no effective tools to visualize and analyze the stacking and topological structures of COFs, further limiting the ability to control the quality and shape of COFs.(2) Hydrolytic and hemodynamic stabilityOwing to the reversible nature of the linkages in the COFs, these crystalline porous polymers often suffer from limited stability, especially in the humid, acidic and basic environments. On the one hand, these reversible linkages endow the COFs with desirable sensitivity for some special purposes, such as controlled drug release and environment-triggered degradation. One the other hand, the limited stability will markedly reduce the materials service-life time and thus significantly prevent their practical applications. As a result, the synthesis of a stable COFs that is robust against harsh conditions or physiological environments remains a major issue that need to be addressed. The design and development of new linkages for fabrication of COFs may be an effective solution to overcome these issues.After injection or implantation in body, the interactions between the materials with serum proteins, cells and tissues will determine the performance of biomedical materials. Especially for the materials used in drug delivery, they should have the ability to avoid the clearance of the nanocarriers by the reticuloendothelial system and enable the circulation time to overcome the biological obstacles in the path of delivery. Therefore, to achieve high therapeutic efficacy and low side effects, the idea COFs for drug delivery should be able to sufficiently stabilize to tightly retain the drug in the bloodstream and maximize the accumulation in targeted site. However, so far few of COFs can simultaneously meet these requirements. Some of these issues may be effectively overcome by surface modification though inert hydrophilic polymers, such as PEG, or by embedding them in a supporting matrix or substrate, such as hydrogels or liposomes.(3) Biocompatibility and systemic toxicityA detailed and careful assessment of the factors that can influence the biocompatibility and toxicity of COFs is crucial for their safe and sustainable development in the pharmaceutical and biomedical fields. However, although there are several studies performed to test the cellular toxicity of COFs, as a novel kind of nanomaterials, the hemocompatibility, histocompatibility, cytotoxicity and neurotoxicity of COFs are unclear at this stage, let al.one the long term in vivo assessments on the absorption, distribution, metabolism and excretion of COFs. Especially considering polycyclic aromatic compounds as the starting materials of COFs, more studies of systematic toxicity, biocompatibility and biological effects, especially in animal models, are urgent and fundamental issues that require thorough investigation, before COFs-based biomaterials can be applied for human welfare. In this context, the exploration of new compatible building blocks and linkage reactions may be the key to expand the COFs biomedical applications.Although there is still a long way before clinical applications of COFs, we believe the rapid development of these materials will make them become a potential platform for biomedical and pharmaceutical purposes, opening up some new avenues for exciting opportunities to improve human welfare. Undoubtedly, collaborative, multidisciplinary research efforts are still needed for more successful applications of COFs in biomedical science.


Although Zn biofortification increased Zn bioavailability in standard and refined flour, it did not reduce the bioavailability of iron, manganese, or copper in wheat flour.Zinc (Zn) deficiency is a common disorder of humans in developing countries. The effect of Zn biofortification (via application of six rates of Zn fertilizer to soil) on Zn bioavailability in wheat grain and flour and its impacts on human health was evaluated. Zn bioavailability was estimated with a trivariate model that included Zn homeostasis in the human intestine. As the rate of Zn fertilization increased, the Zn concentration increased in all flour fractions, but the percentages of Zn in standard flour (25%) and bran (75%) relative to total grain Zn were constant. Phytic acid (PA) concentrations in grain and flours were unaffected by Zn biofortification. Zn bioavailability and the health impact, as indicated by disability-adjusted life years (DALYs) saved, increased with the Zn application rate and were greater in standard and refined flour than in whole grain and coarse flour. The biofortified standard and refined flour obtained with application of 50 kg/ha ZnSO Zinc (Zn) is one of the most abundant trace elements in human bodies, with 1.5–2.5 g present in the average adult . Zinc deIn more than 22 developing countries, at least 60% of the Zn in human diets is derived from C3 grains and legumes . Wheat iAn important approach to preventing Zn deficiency in humans is Zn biofortification, i.e., the use of agronomic practices, conventional plant breeding, or biotechnology to increase the Zn content of food crops . Rosado In addition to being inherently low in Zn, wheat is rich in phytic acid (PA), a compound that limits Zn bioavailability . Phytic The burden of disease and injury in human populations is usually quantified in terms of disability-adjusted life years (DALYs) . DALYs hMicronutrients such as iron (Fe), manganese (Mn), and copper (Cu) are also essential for wheat growth and human health. Research on the relationship between Zn and other micronutrients in wheat and in humans has produced inconsistent results. Majid et al. , for exaThe objectives of this study were (1) to quantify the effects of Zn biofortification via fertilizer application on Zn and PA concentrations in wheat grain and in different kinds of flours; (2) to estimate the resulting bioavailability of Zn in humans; and (3) to assess the health impact of Zn-biofortified flours. The relationship between Zn biofortification and the bioavailability of other micronutrients was also evaluated.w/v in water) was about 8.0, and the diethylene triamine pentaacetic acid (DTPA)-extractable Zn, Fe, Cu, and Mn concentrations before sowing were 0.45, 5.52, 0.82, and 5.30 mg/kg, respectively. The same winter wheat (Triticum aestivum L.) cultivar  was used in both cropping seasons. Six Zn application rates were assessed: 0, 10, 25, 50, 100, and 150 ZnSO4·7H2O (22% Zn) kg/ha. The experiment had a randomized complete block design with four block and 24 plots; each plot was 75 m2. Before sowing, a compound fertilizer  was applied. Another 150 kg of N/ha was supplied as urea at the stem elongation stage. The plots were irrigated at the pre-wintering, stem elongation, and flowering stages in both cropping seasons. At maturity, wheat plants aboveground were removed by hand from a 6-m2 (2 m × 3 m) area in each plot. About 1 kg of grain from these plants was sampled and rapidly washed with deionized water before milling and analysis.A field experiment was conducted in 2013–2014 and 2014–2015 at the Quzhou Experiment Station  in China. In both cropping seasons, wheat was grown from October to the following June in a winter wheat–summer maize rotation system. The soil pH , and the nutrients in the digested solution were determined by inductively coupled plasma optical emission spectroscopy . PA concentrations in different fractions were determined calorimetrically (at 519 nm) as described by Haug andLantzsch [Wheat samples were milled in a Bühler laboratory experimental mill (MLU-202) according to Approved Method 26-21A . Three bLantzsch .TAZ = total daily absorbed Zn (mg Zn/day); MAXA = maximum Zn absorption; TDZ = total daily dietary Zn (mmol Zn/day); RK = equilibrium dissociation constant of the Zn-receptor binding reaction; TDP = total daily dietary PA (mmol PA/day); and PK = equilibrium dissociation constant of the Zn–PA binding reaction. According to Hambidge et al. [MAX, KR, and KP, have constant values of 0.091, 0.680, and 0.033, respectively. TAZ was based on reference adults consuming wheat flour (300 g/day) as a sole daily source of Zn and phytate [A trivariate model based on Zn homeostasis in the human intestine was used to evaluate Zn bioavailability :(1)TAZ=0e et al. , the par phytate  and was  phytate .The DALYs equation was used to estimate the health burden of Zn deficiency and to assess the health impact of Zn-biofortified flours. The current burdens due to Zn deficiency were calculated based on a recent study in China, which indicated that years of life lost (YLL) was 151 million and years lived with disability (YLD) was 202 million years, resulting in a total DALYs value of 352 million years . The stap < 0.05. Statistical Analysis System software  was used for the statistical analysis.One-way ANOVAs were used for many comparisons; means were separated by Fisher’s protected least significance difference (LSD) test at In both cropping years, Zn concentrations in grain milling fractions were significantly greater in the Zn-biofortified treatments than in the no-Zn treatment, and the increases in Zn concentrations were closely related to the quantity of Zn applied . The Zn The estimated Zn bioavailability in whole flour, coarse flour, standard flour, refined flour, and bran increased with the quantity of Zn applied in both cropping seasons . The estIn all four of the most commonly used flours , the daily Zn intake, the percentage of the recommended intake, and the DALYs saved for both infants and children were progressively increased as the rate of Zn fertilization increased . The redZinc biofortification did not significantly affect the concentrations  and bioa4·7H2O/ha . The only undesirable property of PA is that it limits the availability of micronutrients . Erdal eg et al. , the curg et al. .The chemical forms of Zn and its bioavailability in food influence the amount of Zn absorbed through the gastrointestinal tract and into the bloodstream . For humThe Zn levels in the blood plasma, liver, and tibia of rats fed for 3 weeks on diets based on white wheat flour or whole flour were not significantly different, indicating that the whole flour did not reduce Zn absorption or bioavailability . Accordi4·7H2O/ha) could reduce the current healthy burden (DALYs) of Zn deficiency by more than 20%. Among all the four biofortified flours, the effects of Zn biofortification on the estimated Zn bioavailability and on human health impact (DALYs saved) were greatest for standard flour , and the HarvestPlus program  to assesrd flour . This waUnlike other studies ,27, the In the current study, Zn biofortification increased both the concentration and bioavailability of Zn in wheat grain and flours. Although Zn concentrations in wheat whole flour and coarse flour were high, Zn bioavailability and the calculated health impact  were lower in wheat whole flour and coarse flour than in standard flour and refined flour because of the extremely high concentrations of PA in the bran. In addition, Zn biofortification did not influence the bioavailability of Fe, Mn, and Cu. The results indicate that Zn biofortification of wheat by the application of a Zn fertilizer can substantially increase the Zn bioavailability and health impact of wheat flour.


To determine the effect of computer-aided-detection (CAD) software for automated breast ultrasound (ABUS) on reading time (RT) and performance in screening for breast cancer.Unilateral ABUS examinations of 120 women with dense breasts were randomly selected from a multi-institutional archive of cases including 30 malignant (20/30 mammography-occult), 30 benign, and 60 normal cases with histopathological verification or ≥ 2 years of negative follow-up. Eight radiologists read once with (CAD-ABUS) and once without CAD (ABUS) with > 8 weeks between reading sessions. Readers provided a BI-RADS score and a level of suspiciousness (0-100). RT, sensitivity, specificity, PPV and area under the curve (AUC) were compared.p < 0.001). Sensitivity was 0.84 for CAD-ABUS (95% CI 0.79-0.89) and ABUS (95% CI 0.78-0.88) (p = 0.90). Three out of eight readers showed significantly higher specificity using CAD. Pooled specificity  and PPV  were higher in CAD-ABUS vs. ABUS, respectively, albeit not significantly. Pooled AUC for CAD-ABUS was comparable with ABUS .Average RT was significantly shorter using CAD-ABUS  compared with ABUS  (CAD software for ABUS may decrease the time needed to screen for breast cancer without compromising the screening performance of radiologists.ABUS with CAD software may speed up reading time without compromising radiologists’ accuracy.• • CAD software for ABUS might prevent non-detection of malignant breast lesions by radiologists.• Radiologists reading ABUS with CAD software might improve their specificity without losing sensitivity. In mammographic screening the sensitivity in women with extremely dense breasts is only 61% –4, thus Performing supplemental WBUS with handheld devices has limitations. It is relatively time consuming and difficult to compare to prior examinations. Furthermore, handheld WBUS screening is operator dependent and should therefore be performed by trained sonographists, which consequently requires substantial resources . AutomatA downside of supplemental ultrasound screening is the detection of mammographically occult benign lesions that warrant histological verification , 13, 16,Due to the large number of images in the scan, reading a full ABUS examination can be lengthy and cancers may easily be overlooked . ComputeThe need for informed consent for this study was waived by the institutional review board (IRB).ABUS examinations were performed with ACUSON S2000 Automated Breast Volume Scanner systems . This ABUS system acquires 3D B-mode ultrasound volumes over an area of 154 mm × 156 mm using a mechanically driven linear array transducer (14L5). Adequate depth and focus can be obtained using predefined settings for different breast cup sizes. All ABUS examinations were performed by technicians. To ensure coverage of the entire breast two to five overlapping acquisitions were performed at predefined locations. The number of acquisitions depends on the size of the breasts and the possibility to compress the breasts. Per acquisition 318 slices of 0.5 mm thickness are obtained. A dedicated ABUS workstation reconstructs the transverse slices into a 3D volume that can be read in a multiplanar hanging, also showing sagittal and coronal reconstructions.Cases were selected from a large multi-institutional imaging archive that consisted of 2158 ABUS examinations in 1086 women acquired between August 2010 and February 2015 from screening programmes for women at average, intermediate, and high risk and symptomatic women. For each woman a full-field digital mammography (FFDM) examination was also available.n = 919), “benign” (n = 140), or “malignant” (n = 128) based on radiology and pathology reports from histopathological examinations. “Normal” and non-biopsied “benign” cases were only considered if at least 2 years of negative follow-up was available. Subsequently, from these women with dense breasts, we included all cases with a mammography-negative malignant lesion (n = 20), ten randomly selected malignant cases that were positive on both mammography and ABUS, 30 biopsied benign cases and 60 “normal” cases in the study data set. The study data set thus consisted of 120 unilateral ABUS evaluations, yielding a total of 375 ABUS volumes.The selected cases were anonymised and stripped from information such as age, study date, and imaging institute. All lesions were annotated by a breast imaging researcher with > 3 years of experience with ABUS based on pathology and radiology reports. These annotations served as the ground truth for observer and CAD software detection performance.To select only cases with high breast density, breast density was determined using an automated volumetric software package  on 1657 available unprocessed FFDM images. For 501 examinations, where unprocessed FFDM images were not available, breast density was visually assessed according to the BIRADS lexicon. Examinations of 115 women with a history of breast surgery were excluded; 1187 unilateral examinations of breasts in 715 women were scored as Volpara Density Grade 3 and 4 or BIRADS density categories C or D. We categorised these dense cases as “normal” . In this prototype, each user action was logged with time stamps that were subsequently used to estimate the time spent per case. Commercially developed CAD software  was integrated into this workstation. This CAD software is designed to detect suspicious region candidates in an ABUS volume and mark them with so-called CAD marks Fig. . In addiSeven breast radiologists and one gynaecologist specialised in breast imaging were invited to participate in this study. By inviting readers from different institutes and countries we aimed to increase the applicability of our results to breast imaging practices in different countries, realising that different readers might have slightly varying standards and customs. In some countries, also other clinicians are involved in interpreting breast-imaging examinations. Therefore, we also invited a non-radiologist (gynaecologist) who specialises in breast imaging with approximately 10 years of experience in breast ultrasound and mammography and 8 years of experience with ABUS. Experience with breast imaging for reader one to reader eight was 7, 10, 4, 8, 8, 20, 4, and 20 years and specifically with ABUS was 5, 8, 0, 5, 5, 5, 0, and 0 years, respectively.All eight readers evaluated all cases twice in two separate reading sessions in an independent crossover multi-reader-multi-case (MRMC) study. In each session half of the ABUS cases were read conventionally and half of the cases were read using a CAD-based workflow designed for this study. We counterbalanced the reading modes and changed the case order by randomisation for each reader per reading session. The reading sessions were at least 8 weeks apart  to further minimise any effect of memory bias.Standard ABUS reading was performed in a multiplanar hanging without CAD software. CAD-based reading was performed according to specific instructions of a two-step reading protocol. The first step was to evaluate all CAD marks and dark spots on the MinIP in a case. Subsequently, readers were instructed to scan the coronal reconstruction of each ABUS view in a hanging protocol where coronal reconstructions of all ABUS views of a breast are simultaneously shown.The readers performed a training session of 20 cases to become familiar with the workstation, reading protocol, and CAD software. Readers were given a rough estimate (10-30%) of the prevalence of cancer in the study data set because the criteria for a recall may vary between radiologists who, as in our study, work at different institutes and in different countries  and may In both CAD-based and conventional reading the readers were instructed to mark and rate lesions by placing a finding marker and subsequently determine a BI-RADS assessment score. Because a quasi-continuous linear scale is required to perform receiver-operating characteristic (ROC) analysis, readers were also asked to provide a level of suspiciousness (LOS) score on a scale from 0-100. Note that LOS is not a probability of malignancy as described in the BI-RADS atlas. Instead, readers were recommended to use anchor points referring to the BI-RADS scores with LOS values of 21, 41, 61, and 81 corresponding to the BI-RADS 1/2, 2/3, 3/4, and 4/5 transitions.t-test with 1000 bootstraps to determine the 95% CI and GEE for pooled data. Only the readings recorded within the 95th percentile were included in the analysis to correct for inactivity of the reader during the reading sessions.We determined the sensitivity, specificity, and positive predictive value (PPV) in both reading modes based on BI-RADS scores and compared these parameters per reader using paired McNemar’s and chi-square tests with bootstrapping (1000 samples) to determine the 95% confidence intervals (CI) for individual readers and generalised estimation equation (GEE) for pooled data to correct for repeated measurements. An examination was considered positive if a BI-RADS 3 score  or higher was given. Furthermore, we determined the area under the curve (AUC) and 95 % CI using an alternative free-response receiver-operating characteristics (AFROC) , 20. ForThe ROC analyses were performed using MRMC software . The GEE was performed using the ‘geese’ function in the ‘geepack’ package in R . All other analyses were performed with SPSS statistics 20.0 .Table p = 0.90). Nevertheless, half of the readers detected more cancers with CAD, while only two readers detected fewer cancers using the CAD-based reading protocol. In the CAD-based readings 6 out of 8 readers placed markers on a total of 11 lesions that were actually malignant, but still classified them as benign (BI-RADS 2). In the unaided ABUS reading this happened only in four readers and a total of five malignant lesions. Hence CAD helped in the detection of additional cancers but could not always induce an adequate classification by the readers.Figure p = 0.08). The PPV was on average 13.6% higher for the CAD-based ABUS reading  compared to the conventional ABUS reading  . Overall, seven out of eight readers had higher specificity and PPV with CAD than without. Specificity was significantly higher in three out of eight readers   and this increased to 71% (95% CI 68-75) in the CAD-based reading strategy, although this did not reach statistical significance (p < 0.001)  Fig. , which ip = 0.003), for benign cases by 17.3% , and for normal cases by 16.8%  (p ≤ 0.001).The average reading time for malignant cases decreased by 12.1%  and in dynamic contrast-enhanced breast MRI [maximum intensity projections (MIP)]. Kuhl et al. reported that looking only at MIPs is a reliable and fast (3-30 s per case) approach to breast cancer screening with MRI . The CADOur study has limitations. We did not show corresponding mammograms with the ABUS examinations although these modalities are complementary in most screenings regimes of women with dense breasts and this might positively or negatively affect the screening performance. Furthermore, we enriched the data set with benign and malignant lesions from both screening and diagnostic examinations to increase the power in this study. By doing so, our study data set does not represent clinical practice where the prevalence of benign and malignant lesions is lower. Finally, multiple readers had little experience with ABUS and all readers were inexperienced with the CAD software package that we implemented in our screening environment, which may have negatively affected the screening performance and reading time.In conclusion, our study shows that the CAD software developed for ABUS has the potential to improve the efficiency of reading ABUS by significantly improving the reading speed without decreasing the screening performance. Further research is warranted in a prospective study to investigate the effect of CAD on breast cancer detection, screening recalls, and the interval cancer rate in screening programmes.