P < 0.001 and P = 0.019, respectively). The qualitative and quantitative UWF ICGA image analysis revealed that choroidal blood vessels became dilated, complex, and hyperpermeable as the DR progressed. These features of diabetic choroidopathy (DC) were more severe in eyes with DME than the non-DME eyes.To investigate angiographic characteristic features of diabetic choroidopathy, as well as choroidal vascular density (CVD) and fractal dimension (CFD) in diabetic eyes and controls using ultra-widefield (UWF) indocyanine green angiography (ICGA). All patients underwent UWF fluorescein angiography and ICGA. Using imageJ software, CVD and CFD was analyzed. SFCT was assessed using spectral-domain optical coherence tomography. The image parameters were compared based on the DR stage and the presence of diabetic macular edema (DME). One-hundred six eyes from 63 patients  were included in the DM group, and 40 eyes from 22 subjects were included in the control group. The DM group had a mean age of 59.11 ± 16.31 years and a mean HbA1c percentage of 7.72 ± 1.28%. The most common ICGA findings of DC were choroidal hyperpermeability (57.5%), hypofluorescent spots (48.1%). Salt and pepper pattern (19.8%), inverted inflow phenomenon (3.8%), choroidal arterial tortuosity (24.5%), and late choroidal non-perfusion (6.6%) were more common in advanced DR. The CVD, CFD, and SFCT increased as the DR severity progressed. The DME group had a significantly higher CFD and SFCT than the non-DME group ( Diabetic retinopathy (DR) is the most prevalent and serious microvascular disease induced by chronic hyperglycemia in DM3. This is because the retina has a densely distributed network of blood vessels to meet the high metabolic demand of photoreceptor cells. The choroid, which is located outside the retina, is a unique tissue that is almost made up of blood vessels. It has the most blood flow of any tissue in the human body4. Previous histopathologic studies demonstrated that choriocapillaris degeneration in DM7.Diabetes mellitus (DM) is a chronic metabolic disorder characterized by long-term systemic microvascular and macrovascular complications6. Diabetes caused degeneration of choroidal pericytes and affects CC structure and function10. Recent studies using optical coherence tomography (OCT) angiography indicated that CC loss in DM is highly related to photoreceptor degradation11. Meanwhile, the studies of DC using OCT have focused on subfoveal choroidal thickness, which remains disputed because both thickening and thinning have been detected13. In addition, several studies are limited because they did not take into account the history of ocular treatments and ophthalmologic interventions, such as panretinal photocoagulation or intravitreal anti-vascular endothelial growth factor (VEGF), which clearly have a substantial effect on choroidal thickness17.Diabetic choroidopathy (DC) was previously characterized by choriocapillaris (CC) dropout, luminal constriction, and large and intermediate blood vessel tortuosity18. Previous ICGA image studies show choroidal vascular insufficiency in diabetic patients20. DC displays both hypofluorescent and hyperfluorescent spots in ICGA19. In addition, poor diabetic control was associated “salt and pepper” pattern in non-proliferative diabetic retinopathy (NPDR)20. Recent advances in ultra-widefield (UWF) imaging expand the ICGA’s applicability to evaluate the peripheral vasculature in various retinal and choroidal diseases24. Clinical features of diabetic choroidopathy on ICGA were not yet evaluated using UWF imaging and thus it is needed to be characterized according to the severity of DR. In this study, we analyzed the UWF ICGA findings and association with the choroidal thickness in OCT images to identify choroidal characteristics in ophthalmologically treatment-naïve DM patients.Indocyanine green angiography (ICGA) is a clinically useful imaging technique for assessing the vascular structure of the deep choroid through the retinal tissueIn this study, 106 eyes from 63 patients  were included in the DM group, and 40 eyes from 22 subjects were included in the control group. The baseline characteristics of the studied groups are summarized in Table The most prevalent early and late qualitative DC findings on UWF ICGA were hypofluorescent spots Fig. a,d and cP = 0.009, P < 0.001, and P < 0.001, respectively). In addition, mild NPDR had a significantly lower mean CVD than more severe DR . On the other hand, severe NPDR had a significantly higher CFD than DM with no DR or mild NPDR . In addition, PDR had a significantly higher CFD than DM with no DR, mild NPDR, or moderate NPDR . In DR group, all patients with DME had severe NPDR and PDR. Subgroup analysis of CVD and CFD in severe NPDR and PDR groups based on the presence of DME revealed that the DME group has a significantly higher CFD than the non-DME group, while, CVD had no significant difference between the groups . Sattler’s layer thickness tended to increase according to the DR stage with no significant difference among the groups . Subgroup analysis of SFCT in severe NPDR and PDR groups based on the presence of DME revealed that the DME group has thicker SFCT and Haller’s layer thickness than the non-DME group .Similar to CVD and CFD, SFCT showed a tendency to increase as diabetic retinopathy worsened. Fig. a. PDR haups Fig. b. On theIn this study, we investigated the DC characteristics and quantitatively evaluated the UWF ICGA images in patients with DM. To the best of our knowledge, this is the first UWF ICGA qualitative and quantitative analysis according to DR severity. We demonstrated that hypofluorescent spots and choroidal hyperpermeability were frequent UWF ICGA findings in patients with DC, and the “salt and pepper” pattern, choroidal artery tortuosity, late choroidal non-perfusion, inverted inflow phenomenon were more common in patients with advanced DR. Notably, quantitative image analysis revealed that CVD and CFD were increased according to the DR stage, which was consistent with the increase in SFCT.5. Another histopathologic study, reported the diabetic choroid was characterized by choriocapillaris degeneration, which included increased alkaline phosphatase activity and extravascular migration of polymorphonuclear leukocytes7. These histopathologic studies revealed that DM could affect the choroidal circulation.Diabetic choroidopathy was first described by Hidayat and Fine, which included choriocapillaris dropout, basement membrane thickening, and choroidal neovascularization in patients with advanced DM on light microscopyWith the development of ICGA imaging technology, there have been studies that identified and defined DM.20. They employed conventional 50-degree ICGA imaging and reported a late phase “salt and pepper” pattern and hypofluorescence spots, which is the characteristic pattern of choroidal changes in NPDR. In our study, we discovered a “salt and pepper” pattern is more frequent in advanced NPDR and PDR eyes than in early NPDR or no DR eyes , which are substantially less dense than arteries33. In addition, in recent studies, veins were found to exhibit NG2 and platelet-derived growth factor receptor beta (PDGFRβ) expressing pericytes35. In hyperglycemia, protein kinase C-delta (PKC-δ) upregulation inhibits the PDGF/PDGFRβ-Αkt pathway, reducing pericyte survival and inducing pericyte apoptosis37. Therefore, reduced pericyte coverage or changes in pericyte characters may cause changes in CVD and choroidal thickness in DC.Previous SFCT or subfoveal CVI studies using OCT imaging only show a limited portion of the choroid in the posterior pole. The findings of OCT studies remain controversial. Both choroidal thinning and thickening have been reportedCT Figs. a and 5a.38. The fractal dimension (Df) represents the complexity of the branching pattern of the vasculature, and it is a ratio with no counting units. The reduction in retinal vascular Df indicates early vascular changes in DR40. In this study, CVD and CFD were significantly higher in advanced DR eyes than in early or no DR eyes  is upregulated in diabetic endothelial cells, promoting Smad2/3 signaling in perivascular cells and mesenchymal cell differentiation into VSMC44. TGFβ1 also induces VSMC phenotypic switch to contractile phenotype45. These microvascular mural cell phenotypic changes could be explained by a gradual increase in CFD by the DR stage and an increase in choroidal vascular resistance in DC.A fractal is a geometric pattern that allows the description of objects that branch repeatedly46. Based on the presence or absence of DME, subgroup analysis revealed significantly high CFD, SFCT, and Haller’s layer thickness in the DME group. These findings demonstrate how localized retinal inflammation and VEGF activity in DR affect choroidal vasculature. Based on these findings, CFD with SFCT, particularly Haller’s layer thickness, could be used as a biomarker for treatment response monitoring of DME.DME was caused by a local effect of VEGF and pro-inflammatory cytokines synthesized by the retinaThis study has several limitations. First, the retinal vasculature was not subtracted from the image analysis process. This may result in underestimation of fractal dimension and vascular density in advanced DR with retinal non-perfusion. Second, because images were used without stereographic projection, peripheral CVD may be overestimated. Third, because the UWF ICGA detects mainly medium- to large-sized choroidal vessels, choriocapillaris cannot be analyzed in this study. Fourth, renal function, which has a significant effect on choroidal thickness, was not considered. Finally, because the choroidal vascular disease is ambiguous, the possibility of co-existing choroidal diseases such as pachychoroid spectrum disease cannot be entirely excluded.In conclusion, we revealed that diabetes induces choroidal vascular dilation and increases choroidal vascular complexity through UWF ICGA analysis. An advanced diabetic choroidopathy had a characteristic UWF ICGA pattern, including the “salt and pepper” pattern, inverted inflow phenomenon, late choroidal non-perfusion, and choroidal artery tortuosity, indicating choroidal inflammation and increased vascular resistance. Future longitudinal studies should focus on DC clinical staging and its therapeutic applications.48. Diabetic macular edema (DME) defined as central macular thickness 49. In this study, control group were subjects without DM who visited a retina clinic to assess vitreous opacity, epiretinal membrane.This cross-sectional, retrospective, observational study was conducted at the Ophthalmology Department of Asan Medical Center in the period between June 1, 2020, and February 28, 2021. The study protocol adhered to the principles of the Helsinki Declaration and was approved by the Institutional Review Board and Ethics Committee of the Asan Medical Center. A treatment-naïve patients with DM who underwent UWF ICGA were included in this study. The exclusion criteria were as follows: (1) age less than 18 years; (2) type 1 DM; (3) high myopia  (3200 × 2400 pixel) centered on the fovea was then determined using the Image J oval ROI tool and confirmed by two retina specialists (S.U.C and J.L). The binarized image was then adjusted by Niblack’s auto local thresholding and converted to a red, green, and blue image, and the luminal area was estimated using the color threshold tool. The light pixels were defined as the luminal area, which was automatically calculated by pixel. For CFD, the same ROI, Niblack’s auto local thresholding was used, the binarized image was skeletonized, and the CFD was assessed using the Image J’s FracLac plug-in (https://imagej.nih.gov/ij/plugins/fraclac/fraclac.html).UWF FA and ICGA were performed after obtaining informed consent from the patient. Following an intravenous injection of 5 mL of 10% sodium fluorescein, UWF FA was conducted. UWF ICGA was then performed 30 min after UWF FA, with an intravenous injection of 2 mL of 25 mg of indocyanine green., while choroidal vasculature was quantitatively analyzed using the early phase UWF ICGA images (1–2 min after dye injection). A selected image was converted to a JPG file and the choroidal vascular density (CVD) and the choroidal vascular fractal dimension (CFD) were analyzed using ImageJ version 1.53 h  modified methods from the previous reports  took measurements at a single point below the fovea using the built-in caliper tool .The subfoveal choroidal thickness (SFCT), Sattler’s layer thickness, and Haller’s layer thickness were measured using an EDI SD-OCT image in the subfoveal region. The SFCT was defined as the vertical distance between the hyper-reflective line of Bruch’s membrane and the line connecting the outer margin of the large choroidal vessel layer. Haller’s layer was defined as the outer choroidal vessel layer composed of the choroidal large vesselsThe qualitative data were presented in a descriptive manner by DR stages. The quantitative data (CVD and CFD) were compared between DR stages and the groups studied. All statistical analyses were performed using the Statistical Package for the Social Sciences, version 20 . Continuous variables were presented as mean and standard deviation (SD), while categorical variables were presented as numbers (n) and percentages (%). A two-sided p-value of less than 0.05 was considered significant.Presented at the 38th annual meeting of the American Society of Retina Specialists, 2021. San Antonio, United State of America.Supplementary Information.


The Danshen extract was incubated without HLM, with HLM, or with HLM where the active site of the target enzyme was blocked with a specific competitive probe before ultrafiltration to isolate ligand–enzyme complexes from unbound compounds. Subsequently, HPLC analysis was performed on the released ligands from the complexes. α-Naphthoflavone, ketoconazole, and sulfaphenazole were used as specific competitive probes for CYP1A2, 3A4, and 2C9, respectively. The signal-to-noise ratio (S/N) and specific-signal-to-noise ratio (S-S/N) of each compound were calculated using the obtained peak areas. Finally, two criteria were applied to select putative ligands for each specific target: (1) S/N > 1; (2) S-S/N > 0. Finally, dihydrotanshinone was identified as a specific ligand for CYP1A2 and tanshinone I, cryptotanshinone, and tanshinone IIA were identified as specific ligands for CYP1A2, 2C9, and 3A4. It was demonstrated that the newly developed method can be used to rapidly and directly detect specific ligands from natural product extracts in multi-target systems.A novel  The specific ligands in natural product extracts could be identified from a multi-target system by ultrafiltration-high performance liquid chromatography using competitive probes. To predict the metabolism-based interactions between drugs and herbal products, the components of herbal products as well as their effects on the activities of hepatic drug-metabolizing enzymes should be identified.3,4 Unfortunately, the chemical complexity of herbal products makes understanding the interactions of each component with the related drug-metabolizing enzyme impossible before isolation, regardless of the method used (in vitro or in vivo).Since the use of herbal medicines has become increasingly popular for the treatment of various diseases in the past two decades, the safety of the co-administration of such products with conventional drugs should be evaluated, because herb–drug interactions may occur, which could lead to adverse outcomes through phytochemical-mediated inhibition or induction of hepatic drug-metabolizing enzymes.5 Thus, examination of the HPLC profile of certain peaks can be used to identify “hits”. Affinity-based ultrafiltration-HPLC has significant advantages including rapid and convenient operation, low cost, high hit ratio, and lack of immobilization of screening targets on a carrier as in other active molecular screening methods such as bioassay-guided fractionation, biochromatography, magnetic separation, microdialysis, hollow fiber adsorption etc.6–8 Even though the disadvantages of affinity-based ultrafiltration-HPLC such as false positive results and false negative results can be avoided,9 high concentrations of pure screening targets  are required.10 To our knowledge, only a small proportion of commercially available pure targets  are cheap and stable, and most are expensive; many targets cannot be prepared easily and are not commercially available. The use of tissue products  or cell lysates is more feasible and cost effective. Nevertheless, previous studies have only described multi-ligand screening in single-target systems on affinity-based ultrafiltration-HPLC platforms, and using this method for screening in a multi-target system is still a challenge.Affinity-based ultrafiltration-high performance liquid chromatography (HPLC) has emerged as a rapid and convenient approach for protein–ligand interaction studies, and has been widely applied for the screening and identification of ligands of enzymes, proteins, or receptors from natural product matrices, in which small molecules from natural product extracts bound to selected targets  can be separated from unbound small molecules by ultrafiltration and subsequently identified.Salvia miltiorrhiza) ethanol extracts (DEE), in which human liver microsome (HLM) was used as the source of CYP enzymes instead of pure recombinant human enzymes. The CYP superfamily is an important drug-metabolizing enzyme system and is involved in the biotransformation of a number of exogenous  and endogenous compounds . It is almost universally accepted that over 90% of drug metabolism in humans is mediated by CYP enzymes.11 Moreover, among the CYPs identified to date, enzymes from CYP1, CYP2, and CYP3 families catalyse the biotransformations of the majority of drugs used in clinics today. Danshen is the dried root and rhizome of Salvia miltiorrhiza and is a widely used medicinal plant for the treatment of cardiovascular disease in China and serves as a complementary medicine in the West.12 Among the major active constituents of Danshen that have been isolated and characterized, tanshinones have been reported to exhibit anti-platelet, cardio-protective, anti-inflammatory, hepato-protective, and anti-HIV effects in preclinical studies.13 Moreover, a number of herb–drug interactions leading to adverse outcomes were reported to involve Danshen when it was co-administered with certain therapeutic agents.14,15 As the pharmacokinetics of Danshen have been well studied, it was selected to validate the proposed method.Herein, we propose a novel strategy for affinity-based ultrafiltration-HPLC for the detection of specific ligands of multiple targets from natural product extracts using label-free competitive probes. As a proof-of-concept, the specific ligands of cytochrome P450 (CYP) 1A2, 2C9, and 3A4 were directly identified from Danshen . During the incubation process, ligands in the matrix bind to targets to form complexes, which can be separated from unbound small molecules through the ultrafiltration membrane. The ligands that dissociated from complexes retained on the ultrafiltration membrane were collected and subjected to HPLC analysis to obtain the chromatogram of bound ligands in the mixture. To eliminate non-specific binding induced by interactions between the ligands and ultrafiltration membrane, a blank group was carried out by incubating without the targets mixture before ultrafiltration. To determine the specific ligands, a control group was carried out with incubation in the presence of targets mixture where the active site of the expected target enzyme was blocked with a specific competitive probe before ultrafiltration. According to In order to identify specific ligands for a target enzyme in a multi-target system, a strategy for affinity-based ultrafiltration-HPLC combined with competitive binding was developed. The general procedures are summarized in 16 In the present study, α-naphthoflavone, ketoconazole, and sulfaphenazole were selected as competitive probes for CYP1A2, 3A4, and 2C9, respectively (1A2 = 0.09) was identified as a specific ligand of CYP1A2; compound 2 , compound 3 , and compound 4  were identified as specific ligands of CYP1A2, 2C9, 3A4. The four compounds were identified as dihydrotanshinone (1), tanshinone I (2), cryptotanshinone (3), and tanshinone IIA (4)  .17 but Chen et al. first used competitive binding experiments in ultrafiltration-HPLC to eliminate false positives.18 In fact, not only false positive, but also ligands with different binding sites from competitive binding compounds will be eliminated by such competitive binding experiments. Thus, Song et al. and Wang et al. proposed that competitive binding experiments could be used to identify selective and specific enzyme inhibitors from natural products.9,19 Recently, Wang et al. concluded that competitive binding experiments could reduce the false positive results of affinity-based ultrafiltration-HPLC.20 Alternatively, in multi-target systems, all ligands binding to unexpected targets can be considered false positive results, and the specific ligand of the expected target enzyme might be distinguished by competitive binding experiments by adding a specific competitive probe compound. We proposed the developed method under this assumption. However, one limitation of competitive binding experiments is that not all false positives can be eliminated and some ligand candidates may be excluded, similar to the results obtained herein. Wang et al. indicated that this limitation is due to the properties of the competitive binding compounds, including binding site and affinity differences between ligands and competitive probes.20 Put simply, the developed strategy will only work when binding site of the ligand, which has a similar or lower binding affinity than the competitive binding probe. Thus, selecting an appropriate competitive probe is critical. Dihydrotanshinone was not identified as a specific ligand for CYP3A4 and CYP2C9 perhaps because dihydrotanshinone has a different binding site than the probes or has a relatively high binding affinity towards the target enzyme.To validate our results, we summarized the data obtained in this and previous studies using traditional inhibition methods . The resin vitro kinetic analysis and in silico computer simulation of docking model are presented in Ki = 0.53 μM) and CYP2C9 (Ki = 1.92 μM) and is a non-competitive inhibitor of CYP3A4 (Ki = 2.11 μM); tanshinone I is a competitive inhibitor of CYP1A2 (Ki = 2.16 μM), CYP2C9 (Ki = 51.20 μM), and CYP3A4 (Ki = 86.90 μM); cryptotanshinone is a competitive inhibitor of CYP1A2 (Ki = 1.88 μM), CYP2C9 (Ki = 22.90 μM), and CYP3A4 (Ki = 120.40 μM); tanshinone IIA is a competitive inhibitor of CYP1A2 (Ki = 1.45 μM), CYP2C9 (Ki = 61.60 μM), and CYP3A4 (Ki = 218.70 μM). These results suggested that (1) dihydrotanshinone might be a specific, high affinity ligand of CYP2C9 because it exhibited competitive inhibition and a much lower Ki value than the other three compounds; (2) dihydrotanshinone might be a non-specific, high affinity ligand of CYP3A4 because it showed non-competitive inhibition and a much lower Ki value than the other three compounds. The computer simulation results  and 0.5 M potassium phosphate buffer (pH 7.4) were purchased from Corning . α-Naphthoflavone, ketoconazole, sulfaphenazole, dimethyl sulfoxide (DMSO), and trifluoroacetic acid (TFA) were obtained from Sigma-Aldrich . All organic solvents were of HPLC-grade and were obtained from J. T. Baker . Ultrapure water with a resistivity over 18.2 MΩ cm, generated by a Milli-Q laboratory water purification system , was used for all solutions, dilutions, and HPLC analyses. All other reagents, unless otherwise specified, were purchased from Sigma-Aldrich as were used as received without further purification.HLM  was deposited at the Center for Efficacy Assessment and Development of Functional Foods and Drugs, Hallym University in Chuncheon, Korea. Danshen was extracted 3 times with ethanol for 3 h. The solvent was evaporated under reduced pressure below 45 °C to give the ethanol extract.−1) for further assays. The HLM was dissolved in potassium phosphate buffer  to prepare HLM potassium phosphate buffer stock solution (5 mg mL−1) for further assays. The probes including α-naphthoflavone, ketoconazole, and sulfaphenazole were dissolved in 20% methanol (v/v) to prepare the probe stock solutions (5 Mm) for the competitive binding experiments.The DEE was dissolved in DMSO to prepare the extract sample solution  and were separated from the denatured proteins by centrifugation, and were subsequently washed with 50% methanol (v/v). The released bound ligands were combined and dried under nitrogen gas for 3 h. Next, the dried bound compounds were reconstituted in 50% methanol  and the reconstituted bound fractions were subjected to HPLC analysis using the methods described in Section “HPLC conditions”. The blank experiment was carried out similarly, but without the addition of probe pre-incubated HLM, which was prepared following the methods described in Section “Competitive binding experiments”. To rank the binding affinities of the ligands to HLM, the S/N was calculated using Aa and Ab are the HPLC peak areas of released bound ligands from samples incubated with and without HLM, respectively.30 μL of DEE sample solutions, 6 μL of HLM potassium phosphate buffer stock solution, 30 μL of 20% methanol water solution (v/v), and 234 μL of 0.5 M potassium phosphate buffer (pH 7.4) were mixed and incubated at 37 °C for 60 min. The incubated mixture was filtered through a Microcon YM-10 centrifugal filter unit with a 10 000 MW cut-off membrane (Millipore) by centrifugation at 6350 × −1 HLM potassium phosphate buffer  stock solution was incubated with 30 μL of a 5 mM probe stock solution at 37 °C for 1 h. Then, the probe pre-incubated HLM and extract sample solution  were mixed in a total volume of 300 μL and incubated at 37 °C for 1 h. The incubated mixtures were subjected to ultrafiltration and HPLC analysis following the methods described in Sections “Ultrafiltration procedures” and ‘HPLC conditions”. The S-S/N was defined as the competitive binding ability of specific CYP enzymes in HLM between its specific probe and ligands. S-S/N can be calculated using Aa, Ab, and Ac are the HPLC peak areas of released bound ligands incubated with HLM, without HLM, and with probe pre-incubated HLM.The competitive binding experiments were carried out using the probe pre-incubated HLM. α-Naphthoflavone was used as the competitive probe for CYP1A2, sulfaphenazole was used as the competitive probe for CYP2C9, and ketoconazole was used as the competitive probe for CYP3A4. For the pre-incubation of HLM with each specific probe, 60 μL of a 5 mg mL−1. The optimized gradient chromatography conditions were 5–100% B from 0–105 min. The eluent was monitored at 254 nm.HPLC analysis was performed on a Dionex system  comprised of a P850 pump, an ASI-100 automated sample injector, STH585 column oven, and UVD170S detector. Separation was achieved on an Eclipse Plus C18 column , coupled with a guard column at 30 °C. Briefly, 25 μL of the prepared ligand solution was injected into the system and eluted with acidified water  and methanol (B) at a flow rate of 0.7 mL min2HI4 for CYP1A2; 1R9O for CYP2C9; 2V0M for CYP3A4). The protein preparation wizard in the Sybyl software was used to remove non-protein atoms and water molecules from PDB files, to correct structural defects in the raw structure such as missing atoms and residues, and for energy minimization. The probe and ligand structures were constructed using Sybyl. Prior to docking, the probe and ligand molecules were optimized using the Tripos force field and MMFF94 partial charges. The “thresh” and “bloat” were set to 0.5 and 1 for generation of a protomol, and the number of additional starting conformations and resulting docking poses were set to 5 to 20, respectively. To validate the docking process, the redocking of enzymes with the inhibitors which are originally in the complexes downloaded from RCSB Protein Data Bank were performed, and the root-mean-square deviation (RMSD) for each enzyme was provided in the ESI.We used the Surflex-dock software for molecular docking analysis. The 3D crystal structures of human CYP1A2 (in complex with α-naphthoflavone), 2C9 (in complex with flurbiprofen), and 3A4 (in complex with ketoconazole), solved at resolutions of 1.95 Å, 2.0 Å, and 2.8 Å, respectively, were obtained from the RCSB Protein Data Bank (accession id: in vitro strategy for affinity-based ultrafiltration-HPLC with competitive binding probes for the direct identification of specific ligands in multi-target systems from natural products was proposed. Using CYP1A2, 3A4, and 2C9, specific ligands from Danshen extracts, where HLM was used as the source of CYP450 and α-naphthoflavone, ketoconazole, and sulfaphenazole were used as specific competitive probes, we concluded that the newly developed method can be used for the identification of specific ligands from natural product extracts in multi-target systems.In conclusion, a novel There are no conflicts to declare.RA-008-C7RA12161J-s001


We describe the case of a 1-year-old girl with food protein-induced enteropathy (FPE) that was difficult to diagnose. She was referred to our hospital with a 3-month history of diarrhea, vomiting, and weight loss. Although her diarrhea improved after a few days of fasting, oral intake of elemental diets, formula milk, or rice porridge resulted in repeated relapses. The serum IgE level was 1028 IU/mL, and radioallergosorbent tests were positive for milk, casein, alpha-lactalbumin, and other allergens. A histopathology of the duodenal mucosa revealed loss of mucosal villous structure, crypt hyperplasia, crypt apoptosis, and lymphocyte and eosinophil infiltration (<20 eos/hpf) into the lamina propria. After prednisolone (PSL) therapy and the complete removal of cows’ milk and chicken eggs from her diet, the patient’s diarrhea disappeared. Five months after discontinuing oral PSL and complete removal of cows’ milk and chicken eggs, the duodenum exhibited normal mucosal villous structure and well-differentiated ducts. No abnormalities were observed in the egg rechallenge; however, diarrhea recurred after the cows’ milk rechallenge. Thus, histopathologic examination of the gastrointestinal mucosa is useful for diagnosing FPE similar to oral food challenges, and re-evaluation after elimination diet therapy may be beneficial to rule out other diseases. Non-immunoglobulin E (IgE)-mediated gastrointestinal food allergies (non-IgE-GIFAs) and eosinophil gastrointestinal disorders (EGIDs) are collectively referred to as gastrointestinal allergies. Gastrointestinal allergies are allergic reactions with gastrointestinal symptoms and are classified into three types: IgE-dependent, non-IgE-dependent, and mixed with both properties. Normal immediate food allergies are IgE-dependent, non-IgE-GIFAs are not IgE-dependent, and EGIDs belong to the mixed gastrointestinal allergy group ,2,3,4.Non-IgE-GIFAs are classified as food protein-induced enterocolitis syndrome (FPIES), food protein-induced allergic proctocolitis (FPIAP), and food protein-induced enteropathy (FPE) ,6,7. FPIHerein, we describe an infant case of FPE that was difficult to diagnose. Improvement in the gastrointestinal mucosal pathology after removal of the causative food, based on esophagogastroduodenoscopy (EGD) findings, facilitated the diagnosis.3 cells/mL. Her laboratory test findings were as follows: total protein, 6.4 g/dL (NR: 6.8–8.1); albumin, 3.9 g/dL (NR: 4.1–5.1); aspartate aminotransferase, 35 IU/L (NR: 20–45); alanine aminotransferase, 17 IU/L (NR: 4–24); blood urea nitrogen, 7.4 mg/dL (NR: 8–20); C-reactive protein, 0.47 mg/dL (NR: <0.03); sodium, 135 mEq/L (NR: 137–147); potassium, 3.6 mEq/L (NR3.6–5.2); bicarbonate, 19.1 mmol/L (NR: 21.0–27.0); and lactic acid, 21 mg/dL (NR: 4–16). At this time, a sufficient number of calories, amino acids, and fat was being administered intravenously. The serum IgE level was 1028 IU/mL (NR < 30), and radioallergosorbent tests were positive for milk (63.7 UA/mL class 5), casein (9.8 UA/mL class 3), alpha-lactalbumin (51.6 UA/mL class 5), egg white (58.8 UA/mL class 5), egg yolk (10.8 UA/mL class 3), ovomucoid (13.2 UA/mL class 3), wheat (14.5 UA/mL class 3), and soybean (1.3 UA/mL class 2). Skin prick tests were not performed to confirm the results of specific IgE test. Immunoglobulin (Ig) levels  and lymphocyte subset ratios were normal. Fecal eosinophil tests were negative, and no infection was detected in stool culture tests. Stool adenovirus, rotavirus, and norovirus antigen tests were negative.A 1-year-old girl was referred to our hospital with a 3-month history of diarrhea and weight loss. She was fed a mixed diet (breast and formula milk) from birth, beginning with rice and soybeans at 6 months and progressing to cows’ milk, wheat, and chicken eggs (yolk and white) at 8 months. She did not develop anaphylactic symptoms with formula milk and cows’ milk intake. There were no accompanying signs and symptoms related to other organs, such as mild eczema, skin dryness, or frequent upper respiratory tract infection due to cows’ milk allergy. Neither she nor anyone in her family had allergic diseases, such as atopic dermatitis or asthma. At 9 months of age, she developed vomiting, diarrhea, and anorexia; her previous diagnosis was infectious gastroenteritis and postenteritis syndrome. One month before her transfer to our hospital, she was admitted to the previous hospital because her symptoms did not improve for two months, and her weight loss progressed. During the 2 months prior to her admission, her diet was unrestricted. Although her vomiting improved after a few days of fasting, oral intake of elemental diets, formula milk, or rice porridge resulted in repeated diarrheas. Upon admission to our hospital, her physical examination revealed the following: height, 69.0 cm (standard deviation [SD] −2.2); bodyweight, 6641 g (SD −2.8), 999 g lesser than the weight measured 3 months ago; body temperature, 37.1 °C; heart rate, 96 beats per minute; and blood pressure, 88/50 mmHg. Her blood test results were as follows: white blood cell count, 16,900/mL ; eosinophil percentage, 2.0% (NR: <5.6); hemoglobin, 9.9 g/dL (NR: 13.7–16.8); and platelet count, 679 × 10Imaging and endoscopy examinations were performed to determine the cause of the prolonged diarrhea. Abdominal computed tomography and ultrasound examinations revealed no abnormalities in the small intestine; the large intestine was dilated but no wall thickening was observed, suggesting nonspecific enteritis. An EGD showed no macroscopic abnormalities from the esophagus to the duodenum. However, histopathology of the duodenum mucosa revealed the loss of mucosal villous structure, crypt hyperplasia A,B, crypAutoimmune enteropathy and inflammatory bowel disease (IBD) were suspected; therefore, additional tests were performed. Antiautoimmune enteropathy-related 75 kDa antigen  and antiDiagnosis of non-IgE-GIFAs is difficult for the following reasons: (1) non-IgE-GIFAs are mainly caused by cows’ milk; however, rice, soybeans, wheat, chicken eggs, allergens in maternal diet transmitted through the consumption of breast milk, and other allergens can also cause non-IgE-GIFAs; (2) the causative food cannot be estimated from the specific IgE antibody, as non-IgE-GIFAs is not IgE-dependent; (3) 30% of non-IgE-GIFAs are also positive for specific IgE similar to IgE-dependent immediate food allergies; and (4) no clear diagnostic criteria for FPIAP and FPE exist. In the present case, the specific IgE antibodies were also positive for foods other than milk. The gastrointestinal mucosal findings obtained using EGD before and after elimination diet therapy were helpful in making the diagnosis.Non-IgE-GIFAs may not be fully diagnosed , and theFPE causes diarrhea that lasts for more than 2 weeks, leading to poor weight gain and stunted growth due to digestive and absorptive disorders. The pathologic findings for FPE are well known, and histologic findings such as atrophy of small intestinal mucosal villi, crypt hyperplasia, and lymphocyte infiltration are useful for diagnosis. These features were observed in the present case. In contrast, EGIDs are characterized by extensive eosinophilic inflammation and mast cell infiltration . In the In allergic diseases, removal of the causative antigen facilitates both diagnosis and treatment. Elimination diet therapies have not been established for non-IgE-GIFAs; therefore, it is difficult to assess improvements in gastrointestinal symptoms with these therapies. Elimination of the offending food significantly improves emesis and diarrhea within a few hours in patients with acute FPIES and within days in those with chronic FPIES. Visible blood in the stool resolves within a few days in patients with FPIAP. However, in patients with FPE, symptoms usually resolve in 1–4 weeks . The varWith regard to the present case, we should be careful about her elevated IgE levels when diagnosing FPE. Approximately 30% of non-IgE-GIFAs are known to be positive for specific IgE . In the If a restricted diet method has not been established, pathologic evaluation by EGD in infants may be necessary for distinguishing between allergic and nonallergic diseases. Even though EGD is invasive, the procedure may be necessary for eliminating unnecessary testing and surgical interventions . In conc


Herein, we collected the acute toxicity data of 11 aquatic species from 10 families in 5 phyla to fit the metrical-based SSDs, meanwhile generating the ICE-based-SSDs using three surrogate species , and finally compared the above SSDs, as well as the corresponding HC5. The results showed that the measured HC5 for acute MPs toxicity data was 112.3 μg/L, and ICE-based HC5 was 167.2 μg/L, which indicated there were no significant differences between HC5 derived from measured acute and ICE-based predicted values thus the ICE model was verified as a valid approach for generating SSDs with limited toxicity data and deriving WQC for MPs.Microplastics (MPs) in the water environment pose a potential threat to aquatic organisms. The Species Sensitivity Distribution (SSD) method was used to assess the ecological risks of microplastics on aquatic organisms in this study. However, the limited toxicity data of aquatic organisms made it impossible to derive water quality criteria (WQC) for MPs and difficult to implement an accurately ecological risk assessment. To solve the data gaps, the USEPA established the interspecies correlation estimation (ICE) model, which could predict toxicity data to a wider range of aquatic organisms and could also be utilized to develop SSD and HC This pollution cannot be ignored [Significant evidence shows that Microplastics (MPs) have entered the water environment and threaten the well-being of aquatic species ,2,3. Mic ignored . The fun ignored .5 , for evaluating the contaminant threat to species of interest [Species Sensitivity Distribution (SSD) is an important instrument for developing Water Quality Criteria (WQC). However, due to the complexity of test protocols and species availability, results for certain species were unlikely to be obtained. In response to the data gap, the US EPA developed the interspecies correlation estimation (ICE) model, which is based on a log–log correlation between abundant chemical toxicity values for a series of species , at leasinterest ,18,19,20interest .Oncorhynchus mykiss, Hyalella Azteca, and Daphnia magna), and finally compared the above SSDs, as well as the corresponding HC5. Moreover, the WQC of MPs for aquatic organisms was derived using a battery of toxicity data (11 aquatic species from 10 families in 5 phyla) according to the US EPA guidelines.In this study, we collected the acute toxicity data of 11 aquatic species from 10 families in 5 phyla to fit the metrical-based SSDs, meanwhile generating the ICE-based-SSDs using three surrogate species (The objectives of this study were to (i) derive measure-based and ICE-based WQC for MPs and to (ii) compare measured-based-SSDs with original and improved ICE-based-SSDs for MPs. Our study could provide valuable information on pollution management and environmental risk assessment for MPs in the ambient aquatic environment.http://cfpub.epa.gov/ecotox, accessed on 1 March 2022) and two supplemental online databases, CNKI  and ELSEVIER , were used to obtain MP toxicity data. The data were checked and processed in accordance with EPA requirements in the United States [ECOTOX . To begin with, the values of 48-h LCtandards . The detuctivity  and can https://www3.epa.gov/webice/, accessed on 1 March 2022). In this study, the Web-ICE platform was employed. Through a user-friendly interface, the Web-ICE delivered interspecies extrapolation models for acute toxicity. The following requirements were satisfied with the test results of the database: Fish with an LC50/EC50 of 96 h; most invertebrates with a LC50/EC50 of 48 h; fish weighing 0.1–0.2 g; fish less than 1 months old or less than 30 mm in length. Furthermore, MPs’ toxicological endpoints in fish and invertebrates are limited to those associated with death, such as immobility, respiratory suppression, and fatal impacts.The ICE software and more powerful ICE models for aquatic and terrestrial species were created by the US EPA and made accessible on the internet  to predict toxicity values.Based on geometric means from the measured database, the Web-ICE was seeded with acute toxicity values for 50 or EC50 by log-transformed. Where α and β are parameters, which represent the location (or intercept) and the slope of the curve, respectively. Moreover, we used a two-sample Kolmogorov–Smirnov test (K-S test) to analyze the difference between the predicted data group and the measured data group [Log-logistic, log-normal, and Burr III were usually used to fit SSDs . Based ota group . The mai2 value > 0.6; (6) p-values < 0.01. The statistical parameters were critical to assessing the accuracy of the model [According to the US EPA guidelines, the criteria used to pick the predicted toxicity data are briefly listed as follows: (1) mean square error (MSE) < 0.22; (2) taxonomic distance ≤ 4; (3) cross-validation success rate > 85%; (4) degree of freedom (df) > 8; (5) Rhe model ,35,36.2 value ≤ 0.6, or cross-validation success rate ≤ 85%.By running the Web-ICE program, we obtained 133 predicted toxicity values, and only 19 toxicity data were adopted based on the aforementioned ICE criteria, including amphibians, invertebrates, and fish see . One hunFifty-one toxicity values of different species were predicted by Web-ICE for D. magna. Forty-two invalid data were excluded, and only seven species were effective. They were Thamnocephalus platyurus, Daphnia pulex, Simocephalus serrulatus, Utterbackia imbecillis, Amblema plicata, Megalonaias nervosa, and Margaritifera falcata. For O. mykiss, 9 effective data came from a total of 62 data. For H. azteca, three effective data came from a total of 20 data.Tetraselmis chuii, Pseudokirchneriella subcapitata, and Daphnia magna , while the most sensitive species in ICE-based SSD curves were Amblema plicata, Americamysis bahia, Megalonaias nervosa, Ceriodaphnia dubia, Utterbackia imbecillis, and Daphnia pulex . The most tolerant species were a little different between the two ICE models. The most tolerant species predicted by original ICE models were Salmo trutta, Oncorhynchus tshawytscha, and Oncorhynchus kisutch , while the most sensitive species predicted by improved ICE models were Oncorhynchus tshawytscha, Ictalurus punctatus, Oncorhynchus kisutch, Pimephales promelas, and Carassius auratus . Dyer et al. [SSDs were generated by log-logistic, which was constructed using measured toxicity data and ICE predicted toxicity data from three surrogate species . The cumr et al.  also conr et al. .5 values obtained from ICE- and measure-based SSD were different, the Kolmogorov–Smirnov test indicated that the two SSDs showed no significant difference .In addition, the most sensitive species and the most tolerant species were both predicted by improved ICE models, which showed a much wider range of predictions than the original ICE models ,35,36,375 of MPs from ICE- and measure-based SSD were obtained to be 167.2 μg/L and 112.3 μg/L, respectively, which were similar to those reported in previous studies [In this study, the HC studies ,40. The  studies ,42,43.While there are solid ecological reasons to avoid using non-China specifics in the development of China-specific water quality criteria, considerable new research on the evaluation of ecological features as elements worth protecting may lead to reconsideration of such eliminations. If attributes are protected, the location of the species endemicity is irrelevant. This is crucial to assessing danger at a given location. We envisage the creation and deployment of ICE models that include a wide range of species from throughout the world, providing the most rigorous technique for developing environmental criteria regardless of location.5 obtained from ICE-based and metrical acute toxicity values of MPs for aquatic species to assess the accuracy of ICE-generated SSDs. There was no significant difference between the ICE-based and measured-based SSDs, showing that ICE might be a viable method for predicting acute toxicity data for aromatic chemicals. Furthermore, the collection of toxicity data from experiments usually suffered from a long cycle and high cost. Compared with the experimental measurement, the cost of the prediction via ICE models is fairly low, which would save significant time and expense. Thus, in ecological risk assessment, the ICE models are advocated as a good option.This study compared HC


Given the clinical efficacy of β-lactamsas inhibitors of bacterial nucleophilic enzymes, they are of interestas inhibitors of viral nucleophilic serine and cysteine proteases.We describe the synthesis of penicillin derivatives which are potentMpro inhibitors and investigate their mechanism of inhibitionusing mass spectrometric and crystallographic analyses. The resultssuggest that β-lactams have considerable potential as Mpro inhibitors via a mechanism involving reaction with thenucleophilic cysteine to form a stable acyl–enzyme complexas shown by crystallographic analysis. The results highlight the potentialfor inhibition of viral proteases employing nucleophilic catalysisby β-lactams and related acylating agents.TheSARS-CoV-2 mainprotease (M However, some of the initiallyinvestigated penicillin V derivatives appeared to suppress productpeptide ionization at high inhibitor concentrations, perturbing thereliability of the inhibition results. Therefore, the Mpro inhibition assays were performed using an extended 37mer peptidesubstrate based on the same Mpro self-cleavage site (ALNDFSNSGSDVLYQPPQTSITSAVLQ/SGFRKMAFPS-NH2),15 which was less susceptibleto penicillin inhibitor-induced ion suppression. Substituting the11mer peptide with the 37mer peptide in the SPE-MS Mpro inhibition assays did not affect the IC50 values of reportedselected Mpro inhibitors (Supporting Information Table S1); thus, the 37mer substrate was usedfor subsequent IC50 determinations. The observed high Z-factors (>0.5 for each inhibition plate) indicate excellentSPE-MS assay quality using the 37mer substrate peptide .To enableSAR studies, an initial set of penicillin V derivativeswas synthesized based on the identified penicillin V sulfone benzylester Mitor 10; 1I.15 Halpro inhibition assay was used to investigate theinfluence of structural features of the penicillin V sulfone benzylester (10) on potency. Unlike 10, neithercommercially sourced penicillin V (12) nor its benzylester (13)52 inhibited Mpro efficiently, in agreement with previous results using the11mer Mpro substrate15 (13 with meta-chloroperbenzoic acid (mCPBA) afforded the reported penicillin V(S)-sulfoxide benzyl ester (14),52 which is a less efficient inhibitor than thecorresponding sulfone 10 -configuration of 10 using a reported protocol.53 The resultant (6S)-penicillinV (S)-sulfoxide benzyl ester 15-penicillin V sulfone benzyl ester (16), which was obtained from 15 using KMnO4 as an oxidant, was also less efficient in inhibiting Mpro than the (6R)-isomer 10 -configurationat thepenicillin V C6 stereocenter appears to be preferred for efficientMpro inhibition.Next, the effectof the stereochemistry at the C6 stereocenteron inhibition was investigated by inversion of the . Thepotency of 17 was reduced compared to the β-lactam 10  for efficient Mpro inhibition,the impact of its C2 ester group on potency was investigated. To obtaina set of varied penicillin V sulfone esters, the commercially sourcedpenicillin V potassium salt (18) was initially reactedwith different alkylhalides -sulfoxides 21a–r.Havingidentified important structural features of penicillin V sulfonebenzyl ester , whilethe derivative 20g with two fluorine substituents asthe ortho-positions and one at the para-position is more potent (IC50 ∼ 1.6 μM).It is unclear whether these observations reflect alterations in (hydrophobic)interactions of the fluorinated ester groups with Mpro and/or(less likely) enhanced β-lactam reactivity due to remote electroniceffects.The penicillin V sulfone esters ubstrate 2. The pr20h and 20i, whichbear a relatively bulky and electron-withdrawing trifluoromethyl ornitro meta-substituent, inhibited Mpro -sulfoxide esters 21a–r didnot manifest substantial levels of Mpro inhibition (SupportingInformation Table S2), in accord with theinitial SAR results (The presence of a penicillin C2 benzyl esterderivative is notrequired for efficient M22) to investigate the impact of the C6side chain on Mpro inhibition (23), which was then used in amide bond-formingreactions with an appropriate carboxylic acid using COMU58 as a coupling reagent. The resultant penicillinderivatives 24a–n were oxidized with mCPBA tothe penicillin sulfones 25a–n.A set of penicillin sulfone benzylesters with different C6 amidogroups were synthesized in three steps from commercially sourced (+)-6-aminopenicillanicacid ; however, this substitution did not alter Mpro inhibition substantially  appeared to improve inhibition  appeared toimprove inhibition  did notsubstantially affect Mpro inhibition  resulted in substantially decreasedinhibition  as reported65 and investigated forMpro inhibition using the SPE-MS assay , a penam sulfone that is clinically used as a serineβ-lactamase inhibitor (67 The importance of theC6 dibromo substituents for efficient Mpro inhibition isfurther highlighted by the observation that neither sulbactam (33) nor its benzyl ester derivative (34) displayednotable inhibition -sulfoxide para-nitrobenzylester 32 also inhibited with similar efficiency -configured sulfoxide in the absence of a C6 amido directing group.The combined results suggest that the binding mode and/or mechanismof inhibition of the C6 mono- and dibromo-penicillin derivatives 28–32 differs compared to those of theC6 amido penicillin V derivatives previously investigated ,which is reported to selectively alkylate the active site cysteine(Cys145) versus the other 11 Mpro cysteine residues,15 then incubated with selected penicillin sulfoneester derivatives, i.e., 10, 20q, and 25a  TPCK-blockedMpro, in agreement with a mechanism involving a covalentreaction with the nucleophilic active site cysteine residue Cys145.This mode of inhibition is consistent with the results obtained forthe other penicillin sulfone ester derivatives investigated in thisstudy , while γ-lactam 17 appears to inhibit Mpro via a different mechanism , potentially involving noncovalentbinding.To investigate the site of covalent modification, M and 25a 2D–F. Thepro inhibition by penicillinderivatives, we carried out crystallographic studies and obtaineda structure of Mpro complexed with a penicillin sulfone 20e-derived ligand following cocrystallization . The structure was solved by molecularreplacement using a reported Mpro structure ;68 Supporting Information Figure S7).To investigate the mode of M ID: 6YB7) as a sepro-ligand complex activesite provides clear evidence for active site Cys145 S-acylation via a reaction with the β-lactam ring of penicillin 20e  is positioned to interact with the main chain amino groupof Cys145 and Gly143   3B. Note inter alia by the side chainsof His41, Cys44, Met49, Pro52, Tyr54, and Met165. The C6 side chainphenyl group is positioned to π-stack in an offset manner withthe imidazole side chain of His41 (3.8 Å), which is part of thecatalytic dyad; analogous interactions have been observed with otherMpro inhibitors.71 It could be that theC6 phenoxy ether oxygen, which is important for efficient inhibition3C. Furthhibition3, helps 20e viaC5–S bond cleavage follows an initial covalent reaction ofCys145 with the β-lactam, to give an acyclic enamine/imine.An analogous reaction occurs during serine β-lactamase inhibitionby sulbactam and tazobactam.73 We carried out trialrefinements with both the enamine and imine complexes; analysis ofthe electron density implies the presence of a planar C5–C6bond (penicillin numbering), suggesting the presence of the enamine,but we cannot rule out the additional partial presence of the iminetautomer. The SO2H group formed by opening of the thiazolidinesulfone ring projects toward the side chain of Glu166 (3.8 Å)and the main chain amino group of Ser1 (3.0 and 3.3 Å), the latterbeing the N-terminus of the second protomer making up the functionalMpro dimer  human cathepsinsby monocyclic β-lactams,89 (ii) viral cysteineproteases by spirocyclic β-lactams,90 and (iii) mycobacterial l,d-transpeptidases bybicyclic β-lactams.92 Although there is considerablescope for further optimization, our results highlight the potentialof β-lactams as covalently reacting inhibitors of SARS-CoV-2Mpro and, by implication, other  nucleophilic cysteineproteases, including SARS-CoV-2 PLpro.Antibacterial drugs containinga β-lactam ring are amongthe most successful of all small-molecule therapeutics. Their mechanismof action involves reaction with a nucleophilic serine in bacterialtranspeptidases to afford stable acyl–enzyme complexes.pro and PLpro assays, which monitor protease-catalyzed substrate hydrolysisand/or protease modification and which are suitable for inhibitionstudies.93 The MS-based Mpro assays enabledthe identification of certain β-lactams, notably the penicillinV sulfone benzyl ester 10, as covalently reacting Mpro inhibitors.15 In the currentstudy, we report SAR studies that show the potency of 10 can be optimized by about 10-fold, i.e., from IC50 ∼6.5 μM for 10 to IC50 ∼ 0.6 μMfor 28, 31, and 32 -configured penicillin sulfoxides donot inhibit efficiently (R)-configuredsulfoxides do inhibit (20e (R)-configuration is preferred over the (6S)-configuration and benzyl esters being potent inhibitors was less active than the analogous penicillin 10  as a substrate rather than an 11mer peptide (TSAVLQ/SGFRK-NH2). The 37mer peptide was synthesized by solid-phase peptidesynthesis as a C-terminal amide and purified by GL Biochem (Shanghai)Ltd. . Recombinant SARS-CoV-2 Mpro wasprepared according to established procedures;15 note that fresh aliquots, which were not frozen more than once,were used for inhibition assays. Solutions of the inhibitors (100%DMSO) were dry-dispensed across 384-well polypropylene assay plates(Greiner) in an approximately 3-fold and 11-point dilution series(100 μM top concentration) using an ECHO 550 acoustic dispenser(Labcyte). DMSO and formic acid were used as negative and positiveinhibition controls, respectively. The final DMSO concentration waskept constant at 0.5%v/v throughout all experiments (usingthe DMSO backfill option of the acoustic dispenser). Each reactionwas performed in technical duplicates in adjacent wells of the assayplates, and assays were performed in at least two independent duplicates.SPE-MS Mpro (0.3 μM) in buffer ,was dispensed across the inhibitor-containing 384-well assay plateswith a multidrop dispenser (Thermo Fischer Scientific) at 20 °Cunder an ambient atmosphere. The plates were subsequently centrifuged and incubated for 15 min at 20 °C. Note thatwe previously incubated Mpro with inhibitors for 30 or60 min, resulting in more efficient inhibition.15 The substrate mixture (25 μL/well), containing ALNDFSNSGSDVLYQPPQTSITSAVLQ/SGFRKMAFPS-NH2 (4.0 μM) in buffer ,was added using the multidrop dispenser. The plates were centrifuged, and after incubating for 6 min, the reaction wasstopped by addition of 10%v/v aqueous formic acid (5 μL/well).The plates were then centrifuged  and analyzed byMS.In brief, the Enzyme Mixture (25 μL/well), containing Mv/v aqueous formic acid to remove nonvolatile buffer salts. The peptide was eluted from the SPE cartridgewith 0.1%v/v aqueous formic acid in 85/15v/v acetonitrile/water into the mass spectrometer ,and the SPE cartridge was re-equilibrated with 0.1%v/v aqueousformic acid . The mass spectrometer parameterswere as follows: capillary voltage (4000 V), nozzle voltage (1000V), fragmentor voltage (365 V), gas temperature (280 °C), gasflow (13 L/min), sheath gas temperature (350 °C), and sheathgas flow (12 L/min). For data analysis, the m/z +3 charge state of the 37mer peptide (substrate) and the m/z +1 charge state of the SGFRKMAFPS-NH2 C-terminal product peptide were used to extract and integrateion chromatogram data using RapidFire Integrator software (Agilent).Data were exported into Microsoft Excel and used to calculate the% conversion using the equation: % conversion = 100 × /. Normalized dose–responsecurves (formic acid and DMSO controls) were obtained from the rawdata by nonlinear regression (GraphPad Prism 9) and used to determineIC50-values. For compounds 15, 16, 25g, 25c, 29, and 30, the 11mer peptide was used as the substrate.MS analyses were performed using a RapidFire RF 365 high-throughputsampling robot (Agilent) attached to an iFunnel Agilent 6550 accuratemass quadrupole time-of-flight (Q-TOF) mass spectrometer operatedin the positive ionization mode. Assay samples were aspirated undervacuum for 0.6 s and loaded onto a C4 solid-phase extraction (SPE)cartridge. After loading, the C4 SPE cartridge was washed with 0.1%pro (2.0μM) in buffer , was dispensed across thepenicillin-containing 384-well assay plates with a multidrop dispenser(Thermo Fischer Scientific). The reaction mixture was incubated for45 min at 20 °C under an ambient atmosphere prior to analysisby SPE-MS.Solutions ofthe inhibitors (100% DMSO) were dry-dispensed across 384-well polypropyleneassay plates (Greiner) (for 11 or 33 μM top concentrations)using an ECHO 550 acoustic dispenser (Labcyte). DMSO was used as anegative control. Each reaction was performed in technical duplicates.The enzyme mixture (50 μL/well), containing Mpro inhibition, Mpro (2.0 μM) was incubated with the selective Cys145-alkylatingagent TPCK15 (10 μM) in buffer  for 3 h at 0 °C. The mixture was then dispensedacross the penicillin-containing 384-well assay plates with a multidropdispenser (Thermo Fischer Scientific) and incubated for 1 h at 20°C under an ambient atmosphere prior to analysis by SPE-MS.To investigate the importance of the covalent modificationof the active site Cys145 for Mm/z range 850–1350 Da,with a resolution of 2 Da and with a 10–60 kDa cutoff usingthe MaxEnt1 function in Agilent MassHunter Version 7. The deconvolutedfiles were extracted as csv files, sorted using Enthought Canopy GUI,and normalized and plotted using GraphPad Prism 9.MS analyses were performed using a RapidFire RF 365 high-throughputsampling robot (Agilent) attached to an iFunnel Agilent 6550 accuratemass Q-TOF mass spectrometer using a C4 cartridge and the same parametersas described above, with the exception of the gas temperature thatwas reduced to 225 °C. Protein spectra were deconvoluted forthe pro solution was thawed and dilutedto 6 mg/mL . β-Lactam 20e was added to theprotein solution to a final concentration of 10 mM; the mixture wasincubated for 2 h at ambient temperature prior to dispensing the plates.The drop composition was: 0.15 μL protein–ligand solution,0.3 μL 11%v/v PEG 4000, 0.1 M MES, pH 6.5, and 0.05μL Mpro crystal seed stock. A Mpro crystalseed stock was prepared by crushing Mpro crystals witha pipette tip, suspending them in 30% PEG 4000, 5%v/v DMSO,0.1 M MES pH 6.5, and vortexing for 60 s with approximately 10 glassbeads . The reservoir solutionwas: 11%v/v PEG 4K, 5%v/v DMSO, 0.1 M MES, pH6.5. Crystals were grown using the sitting drop vapor diffusionmethod at 20 °C and appeared within 24 h, reaching full sizewithin 36 h. Crystals were looped after 1 week.A frozen SARS-CoV-2 M97 via Xia298 and Aimless99 within CCP4i2.100 The datasets were phased using Molrep101 and the Mpro apo structure (PDBID: 6YB7). Ligandrestraints were generated using AceDRG.102 Typically, 97% of residues are in the favored regions of the Ramachandranplot, 2% in the allowed region, and 1% in high-energy conformations(2 residues). Crystal structures were manually rebuilt in Coot andrefined using Refmac,103 Buster,104 and PDB_Redo (Supporting Information Table S3).105Diffractiondata were collected on beamline I0-3 at the Diamond Light Source at100 K using a wavelength of 0.9762 Å. Data were processed usingDialspro:20e-derived complex have been deposited in the Protein Data Bank (PDB)with accession code 7Z59.The crystal structure data for SARS-CoV-2 M


The use of multi-site datasets in neuroimaging provides neuroscientists with more statistical power to perform their analyses. However, it has been shown that the imaging-site introduces variability in the data that cannot be attributed to biological sources. In this work, we show that functional connectivity matrices derived from resting-state multi-site data contain a significant imaging-site bias. To this aim, we exploited the fact that functional connectivity matrices belong to the manifold of symmetric positive-definite (SPD) matrices, making it possible to operate on them with Riemannian geometry. We hereby propose a geometry-aware harmonization approach, Rigid Log-Euclidean Translation, that accounts for this site bias. Moreover, we adapted other Riemannian-geometric methods designed for other domain adaptation tasks and compared them to our proposal. Based on our results, Rigid Log-Euclidean Translation of multi-site functional connectivity matrices seems to be among the studied methods the most suitable in a clinical setting. This represents an advance with respect to previous functional connectivity data harmonization approaches, which do not respect the geometric constraints imposed by the underlying structure of the manifold. In particular, when applying our proposed method to data from the ADHD-200 dataset, a multi-site dataset built for the study of attention-deficit/hyperactivity disorder, we obtained results that display a remarkable correlation with established pathophysiological findings and, therefore, represent a substantial improvement when compared to the non-harmonization analysis. Thus, we present evidence supporting that harmonization should be extended to other functional neuroimaging datasets and provide a simple geometric method to address it. Functional magnetic resonance imaging (fMRI) has become one of the leading methods to conduct research on human brain mapping. fMRI acquisitions allow the discovery of brain activation patterns, which help to understand the brain processes behind cognition or task performance. A relevant paradigm of acquisition is called resting-state fMRI, where the subject's brain is studied without performing any specific task. Resting-state fMRI made possible to identify intrinsic functional connectivity and resting-state networks in the brain  matrices, which are symmetric matrices with all eigenvalues strictly greater than zero. This set of matrices does not form a vector space. This property implies that some conventional Euclidean operations on SPD matrices, such as subtraction of two SPD matrices, do not yield another element of the set of SPD matrices. Even though one can perform Euclidean operations with them, resulting matrices do not take into account the underlying geometry of the space of SPD matrices. Mathematically, the space of SPD is what is known as a manifold. Roughly speaking, a manifold is a curved space that locally looks like a flat (Euclidean) space. Although one cannot perform Euclidean operations with elements (points) of the space, there exists an extremely powerful mathematical formalism, known as Riemannian geometry, that deals with manifolds and allows computations respecting their underlying geometry. Since functional connectivity matrices are SPD matrices, it is preferable to perform operations on them that take into account their underlying geometry, rather than applying standard Euclidean methods  from multi-site functional connectivity matrices. To this aim, we will adapt two previous contributions to domain adaptation of functional data and we will also propose a method from our own, called Rigid Log-Euclidean Translation. These operations will be particularized to ADHD-200  from four different sources: 20 ADHD/20 HC from Kennedy Krieger Institute (KKI) (Johns Hopkins University), 20 ADHD/20 HC from the NeuroIMAGE sample (NIM), 20 ADHD/20 HC from New York University Child Study Center (NYU), and again 20 ADHD/20 HC from Peking University (PKG). Therefore, we have worked in total with 160 functional connectivity matrices, with dimensions of 190 × 190.n × n symmetric matrix Σ to be positive definite, xTΣ x > 0 for all non-zero x ∈ ℝn. The set of SPD matrices forms a manifold, a topological space that locally looks like Euclidean space. At each point p of some manifold p, which is a vector space and approximates the manifold in the neighborhood of p. In our present case, n × n SPD matrices, which we will denote by n × n matrices, Symn.Symmetric positive definite (SPD) matrices are the set of symmetric matrices with all eigenvalues strictly greater than zero. This property is equivalent to the requirement that, for an g at every point p and the logarithmic map logp at some point p(v) results in a projection of v onto the neighborhood of p in the manifold v. The logarithmic map performs the inverse operation, mapping points in the manifold to vectors in the tangent space of p.Riemannian geometry provides a framework that allows one to regard the manifold as a metric space. Specifically, a Riemannian manifold is a smooth manifold equipped with a SPD tensor field called metric tensor. The metric tensor X, Y ∈ Symn (two symmetric matrices):−1 is the matrix inverse and Tr(·) is the usual trace operator. Associated to this metric, the exponential map at Σ acting on a tangent vector V ∈ Symn and the logarithmic map at Σ acting on a point A) and Log(A) the exponential and logarithm of matrix A, respectively, which can be computed after eigenvalue decomposition A = UDUT as Exp(A) = U exp(D)UT, Log(A) = U log(D)UT, where U is the eigenvector matrix and D the diagonal eigenvalue matrix, and exp(D) (log(D)) denotes the application of the exponential (logarithmic) function to the eigenvalues. A. Finally, under the AIRM framework, the geodesic distance between two SPD matrices Σ and Π isA. Arsigny et al. .Compute Fréchet mean T iterations, perform permutations of the subjects of sizes N+ and N− as previously described, recompute group Fréchet mean, and record the value of the statistic at iteration t, For ij, compute its corresponding p-value by taking the ratio between the number of iterations giving a statistic greater than or equal to T.For each entry The geometry-aware algorithm proposed in You and Park  and implp-values lower than α. It is worth noting that the algorithm tests for statistical significance in absolute difference and, therefore, cannot provide information on the positive or negative character of the difference. The output of the algorithm in our case will be a 190 × 190 matrix with p-values as entries. Nevertheless, we will not rely on a single permutation test on the 80 ADHD/80 HC subjects to perform our analyses. Instead, we have decided to perform 100 separate permutation tests on 10 ADHD/10 HC randomly sampled subjects in each experiment. The reasons for following this approach are first, taking into account the philosophy behind permutation testing, performing a reliable permutation test on 80 ADHD and 80 HC is not computationally tractable, since the number of ways of dividing 160 subjects into two separate groups of 80 are 160!/(80!80!) ~ 1047 and, therefore, by no means one can sample a large enough fraction of permutations to rely on the obtained distribution of statistic values; second, by randomly pooling 10 ADHD/10 HC 100 times, regardless of imaging site, one can probe different combinations of site members and site influence on the variability of the results; and third, one can assess the relevance of salient connections and nodes by checking for coincidences between independent permutation tests. Taken together, this treatment allows evaluating the power of harmonization methods, in the sense that when data is harmonized, one expects more sensitivity  in statistically significant results when using randomly sampled subjects from different sites. Given these aforementioned points, we have operated in the following manner: we have performed 100 separate permutation tests, each one of 1,000 iterations, on 10 ADHD/10 HC randomly sampled subjects; after each test, we have identified the entries with p < 0.001 and declared them as significant; at this point, we have been left with 100 binary matrices of dimensions 190 × 190 (one for each separate test) with ones in the location of significant entries and zeros elsewhere; and, finally, we have summed these binary matrices to obtain a symmetric 190 × 190 matrix, which we call F, with Fij being the number of times the connection ij has been declared significant, that could be regarded as a 2-dimensional histogram.At this point, assuming some confidence level α, one can reject the null hypothesis for entries with Fij = 1), significance is compromised. However, for entries Fij ≥ 2, the (complementary cumulative) probability is ≈ 0.005, and for entries Fij ≥ 3, the resulting (complementary cumulative) probability is ≈ 0.00015. As a consequence, one can threshold the histogram matrix Fij at different frequencies depending on the confidence that one wants to assume.When working at a significance level of α = 0.001 for each individual permutation test, we are assuming that, at most, the probability of mistakenly declaring an entry as statistically significant by chance is 0.001 (incorrect rejection of the null hypothesis). However, when performing 100 separate experiments and considering jointly their results (as in the frequency histograms), this effect can be accumulated. As a simplification, we can model each separate experiment as a random Bernoulli experiment: the probability of mistakenly declaring each entry as significant by chance in a separate experiment is 0.001. The concatenation of several independent Bernoulli experiments follows the binomial distribution. Therefore, we can use the binomial distribution to compute the probabilities for each separate entry to be declared significant by chance once, twice, 3 times, etc. In our case, considering a probability of 0.001 and 100 trials, the binomial distribution predicts that in the final frequency matrix, the probability for each entry to appear once is 0.09. That is, each entry has a probability of 9% of appearing once in the frequency histogram by chance, which indeed means that at this frequency level  was defined as the ratio between the number of entries such that Fij ≥ n and the number of entries such that Fij ≥ 1.Finally, using these notions and taking into account that the previously described procedure will be applied to different imaging-site harmonization methods introduced in Section 2.4, we define a simple quantity that measures the power of a method to specifically point to some entries by accumulating counts across the 100 separate experiments. We call this measure the sensitivity of the method. At a given frequency As mentioned in the Introduction, imaging site has an impact on the acquisition of functional data and their analyses. We show that this is indeed the case for ADHD-200, even when using the same preprocessing pipeline , that is, an invertible n × n matrix. The following properties hold when considering the AIRM framework  = d.The geodesic distance (6) between two SPD matrices Σ and Π is invariant under Σ → Given some Fréchet mean Suppose our dataset can also be divided into collections of matrices Transformation (11) can be expressed as In Ng et al. , authorsC would be different depending on the condition of the subjects. Distances among HC on one side and distances among patients with ADHD on the other side would be preserved, while distances between HC and patients with ADHD would not. This fact could be regarded as the introduction of an uncontrolled source of variability between conditions. On the other hand, it would be desirable to have a harmonization approach that does not depend on the availability of knowledge about subjects' conditions, which would be required when using different transformations. For example, as stated in the Introduction, domain adaptation has been proven useful when training classifiers. Ideally, when the classifier is reliable enough, one would like to assign the condition to a subject without knowing it a priori. Therefore, the use of a single transformation would allow the harmonization of the data prior to the use of the classifier.One could also consider the use of two different Elements from two different tangent spaces cannot be directly compared. To compare them in an appropriate manner, one needs to perform what is called parallel transport. In short, parallel transport refers to transporting a tangent space at a point along a geodesic distorting vectors of this tangent space as less as possible. In Yair et al. , the autk to the tangent space of their site Fréchet mean via logarithmic map Project matrices from a given site 0.Parallel transport them to the tangent space of a common reference point Σvia exponential map exp0Σ(·).Reproject the parallel transported matrices back to the manifold The main line of reasoning applied to our case is as follows:One of the main results of Yair et al. , is that0. Ng et al. . In this case, I , arising the same previously mentioned concerns about the original location in the manifold. On the other hand, Yair et al.  SPD matrices We hereby propose a method for site harmonization, which we call Rigid Log-Euclidean Translation (RLET), that has the two a priori desirable properties stated above: preservation of intra-site geodesic distances, thus retaining biological variability, and consideration of the original location of correlation matrices in the manifold. Site bias could also impact the distribution of matrices around the site mean, but this would require modeling site effects on the distribution of matrices around their mean. The proposed method is therefore a geometrically-motivated first order approximation. We will work under the LERM framework, which allows a purely Euclidean treatment of the logarithm of the matrices. The steps of Rigid Log-Euclidean Translation are as follows:n×n, the above transformation is merely a translation of the original matrices such that transformed site means n×n. These properties are formally proven in the When taking into account that after applying the logarithmic map to the original matrices, we obtain vectors of the Euclidean space ℝI, by removing the term I). RLET(I) can be useful to compare different harmonization strategies at identity and to probe differences coming uniquely from manifold location.Since geodesic distances under the LERM framework are these Euclidean distances, intra-site geodesic distances are invariant under the proposed transformation, and the Rigid Log-Euclidean Translation approach preserves intra-site variability. The transformation also allows retaining information about the original location in the manifold through the global mean. On the other hand, one can straightforwardly modify this approach to rigidly transport matrices to ,∀k, see  and resud = n(n + 1)/2 = 190(190 + 1)/2 = 18, 145). Therefore, the extraction of insights from this space is a challenging task. However, since our manifold is equipped with a metric and a distance measure , we can construct a useful object that can summarize important geometric information: the pair-wise distance matrix Dij = d between functional connectivity matrices (subjects). In fact, this approach of using pair-wise distances or pair-wise similarities is common in unsupervised techniques of dimensionality reduction for visualization purposes, such as multidimensional scaling (MDS) or t-distributed stochastic neighbor embedding (tSNE).Even though all the concepts presented above are geometrically motivated, the major difficulty for bias characterization stems from the fact that we are dealing with a very high-dimensional space , since it was the method of choice in the previous works we have adapted for site harmonization. tSNE  that retain information on the high-dimensional distribution of the matrices in the manifold. Specifically, we have computed pair-wise distances for original (non-modified) matrices I)), We have constructed the aforementioned pair-wise distance matrices distance .dentical . This apI)-harmonized matrices have not been used since, by construction, their pair-wise distances are identical to the ones for RLET(I) matrices compared to RLET matrices, RLET(I) and MW, the identity matrix I. To check that the first property is fulfilled, we have computed pair-wise distance difference matrices I) giving the same results by construction) and I) and RLET(I) , and the imposition of a common Fréchet mean. In the case of RLET . After hI)-harmonized, and MW-harmonized matrices . Therefore, we have obtained four frequency matrices The process of performing 100 separate experiments, described in Section 2.3, to detect entry-wise differences, has been applied, with the same randomly pooled subjects, to the original, RLET, with the matrix being symmetric since we do not have a sense of directed connection; afterward, we have normalized each row , excluding diagonal elements, by using the total number of inter-network anomalies that involve the component represented by that row (DMN); finally, in the last step, we have normalized the diagonal by dividing each diagonal entry by the sum of all diagonal entries . At this point, we are left with a matrix where each row, excluding diagonal entries, adds up to 1, and in turn, diagonal entries add up to 1. One has to interpret this matrix in the following manner: when taking, for example, the row corresponding to DMN, the entry DMN-FP is the percentage of inter-network anomalies involving DMN that end up connected to FP; when considering DMN-DMN, it is the percentage of intra-network anomalies that are found within DMN. Therefore, we have summarized in a plot, the relative contribution of every functional component to the inter-network anomalies involving another specific functional component. One has to bear in mind that DMN-FP (row-column) does not have the same interpretation as FP-DMN and, indeed, their values are different in general : the number of anomalous connections between DMN and FP is fixed, but their relative contribution to the total number of inter-network anomalies involving DMN and to the total number of inter-network anomalies involving FP need not be the same.This 2-dimensional plot is a 10 × 10 matrix  constructed as follows: first, we have filled the matrix by assigning to each entry I)-harmonized, and the MW-harmonized functional connectomes (Fij ≥ 2 to have a large enough number of samples (I) and MW, and between RLET(I); afterward, we have binarized the resulting difference plots at an absolute entry-wise difference ≥ 15%, thus allowing the detection of important changes in the distributions and assessing harmonization and manifold location effects. These results are shown in We have plotted these matrices for the anomalies obtained using the original, the RLET(nectomes . They ha samples . FurtherI) signal distortions induced by relocating matrices to the neighborhood of I, since relative positions between matrices in Log-Euclidean space are exactly the same by construction. Therefore, in the following, we will focus on the comparison between non-harmonized and RLET is the harmonization method that performs the worst  and MW, and the global mean Regarding the detection of significant differences, in terms of sensitivity, RLET and MW (only 3/100 entries display absolute differences larger than 15%), we can claim that harmonization is indeed working in its task of homogenizing results. However, taking into account that the only difference between RLET(I) and RLET(k) so that different sites display approximately the same dispersion around k) and the mean of intra-site-k distances.Finally, given the results pointing to a considerably large intra-site bias, we can propose a modification to RLET circuits, or pair-wise relevant connections. This interpretation makes it possible to probe the fine-grained pathophysiology of ADHD from a functional point of view and to correlate our findings with established results.Although there is a growing consensus that large-scale brain networks are involved in ADHD, the number of anomalous connections that remain as significant when thresholding their frequency at via the striatum, to be reprojected back to the cortical region. Two of these loops play a key role in ADHD: the dorsolateral prefrontal circuit, with the dorsolateral prefrontal cortex (DLPFC) and the  caudate nucleus forming the cortico-striatal pathway, and the orbitofrontal circuit, composed of the orbitofrontal cortex (OFC) and the  caudate nucleus pathway. The dorsolateral prefrontal circuit is a central element in cognitive control, and the orbitofrontal circuit is heavily implicated in reward processing, whose impairment is thought to provoke impulsive behavior in ADHD. On top of these ones, another component that has been added more recently to ADHD PFC anomalous connections is the fronto-cerebellar circuitry. The cerebellum is thought to be involved in diverse functional impairments of patients with ADHD, such as working memory, attention, or the construction of temporal expectations. For a detailed description of the involvement of these circuits in ADHD, we refer the reader to Durston et al. . The motivation underlying the hypothesis of an involvement of PFC is that one of its most important functions is behavioral control, the principal impairment presented by patients with ADHD. The circuitry that has been found to be critical in the neurobiology of ADHD is the cortico-striatal-thalamo-cortical (CSTC) loops. These closed loops transmit cortical inputs to the thalamus n et al.  and refeRemarkably, the analysis of RLET with right superior frontal gyrus (DMN), right postcentral gyrus (MOT) with right lateral occipital cortex superior division (DMN), right occipital pole (VIS) with right lateral occipital cortex superior division (FP), and right lateral occipital cortex superior division (DMN) with right supramarginal gyrus anterior division (DA). Interestingly, 3 out of 4 feature the same brain regions, the right lateral occipital cortex superior division, being associated with DMN and FP functional networks, depending on the particular node of the region.Given the coherence between the results obtained after RLET(In conclusion, we have collected evidence that supports further research on multi-site dataset harmonization when using functional connectivity matrices. Specifically, we have shown that the distribution of matrices in the SPD matrices manifold is site-biased. Moreover, we have been able to discard two adapted domain adaptation approaches and prove that our proposal (RLET(https://doi.org/10.6084/m9.figshare.16437534.v1.The datasets presented in this study can be found in online repositories. The names of the repository and accession number can be found below: Figshare, GS performed the study conception and design, data analysis, code implementation, and material and figures preparation. The first draft of the manuscript was written by GS. DP, GP, and OC provided methodological guidance according to their role of master thesis' supervisors and senior authorship, and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.