SM,|ρ| phasing algorithm has been adapted to the determination of anomalous scattering substructures from single-wavelength anomalous diffraction (SAD) data of protein crystals and successfully tested on data sets mostly retrieved from the Protein Data Bank.The novel  SM,|ρ|) incorporating the inner-pixel preservation (ipp) procedure  has been adapted to the determination of anomalous scattering substructures and its applicability tested on a series of 12 representative experimental data sets, mostly retrieved from the Protein Data Bank. To give an idea of the suitability of the data sets, the main indicators measuring their quality are also given. The dominant anomalous scatterers are either SeMet or S atoms, or metals/clusters incorporated by soaking. The resulting SAD-adapted algorithm solves the substructures of the test protein crystals quite efficiently.Owing to the importance of the single-wavelength anomalous diffraction (SAD) technique, the recently developed |ρ|-based phasing algorithm ( Knowledge of the anomalous scattering (AS) substructure provides starting phase values which can be iteratively improved by density modification. Although the substructure can be solved in favourable cases by the direct interpretation of the anomalous Patterson function  between pairs of acentric reflections (Bijvoet pairs) which follows from the atomic scattering factor definitionj, and N atoms with NA of them scattering anomalously and with r being the atomic position vector. The structure factor of an arbitrary H reflection is thenImportant present applications of the single-wavelength anomalous diffraction (SAD) technique are the location of SeMet atoms in crystals of multi-site genetically engineered proteins, the determination of the positions and occupancies of the heavy atoms (or clusters) entering the crystal, H and −H reflections constituting a Bijvoet pair b)i.e.For two +et al.  phasing serial crystallography studies at synchrotron sources can be found in Nass et al. (2020Equation (6) al. 1989 with therick 2018 and Shelrick 2018. More re al. 2020.SM,|ρ| phasing algorithm  procedure . The aim of the present contribution is the adaptation of the ipp-improved SMAR to the solution of AS substructures from SAD data (SAD-SMAR). Its feasibility is shown with SAD data sets either kindly supplied by the respective authors or retrieved from the Protein Data Bank (PDB). All calculations have been carried out with a modified version of XLENS_v1  The size of the anomalous signal (Bijvoet ratio), et al., 2008(ii) The precision of In SAD phasing, redundancy of diffraction data is an important data collection parameter, since it affects the variance of the average intensity estimates. As this work is based on published data sets, the cited redundancy values are those given by the respective authors.2.D| values, |D|exp, since not all of them are appropriate. A preliminary check should ensure that the Bijvoet-pair reflections have |F|av values satisfying conditions (7a)b)D| differences only those reflections with |F|av values (expressed as |E|’s) larger than a given ECUT cut-off value. In the test calculations, the used ECUT is D| anomalous differences (to increase their reliability and the absence of outliers). Since |D| is in general much smaller than |F|av, random errors inherent to |F+| and |F−| seriously affect the precision of |D|. Consequently, only those reflections fulfilling the |D| > DFCUT × σ(|D|) criterion are preserved in the |D| set  greater than ∼4.0 are filtered out  = root-mean-square deviation of |D|]. The surviving reflections in the |D| set are generically denoted by H.Solving AS substructures by DM requires a previous selection of the experimental |3.3.1.E| values of the substructure s is the resolution shell corresponding to The SAD-SMAR algorithm uses, instead of the experimentally inaccessible quasi-normalized |E| definition, X2 and On the other hand, according to the |s resolution shell, then If X values, SAD-SMAR also uses modified X values called |Xm|. These are obtained (i) by calculating the M modulus function with X as Fourier coefficients (extending the sum to the H reflections), (ii) by suppressing the negative regions in M, and (iii) by back Fourier transforming the modified M function k is the scaling constant putting i.e. the average intensity of the s shell, can be expressed asB is the overall atomic displacement parameter including vibrational and disorder effects. At this point, for convenience, each K constant and the B parameter can be obtained from a Wilson plot, since for each reciprocal-space shell, both The relation between 3.3.ipp procedure, a simple way of enhancing peakness in Fourier maps, was added . Unlike in non-anomalous SMAR applications where Φh contains the phases of all large reflections (i.e. those H reflections with |E| ≥ 1.00), in the case of SAD-SMAR, Φh only includes the H reflections with X larger than a given XCUT cut-off (here XCUT = 1.00). Since E′′| |sin ψ|, the largest possible value of |E′′| for a given X is E′′|  and −1 (for tσ) with σ2 being the variance of h) and t ∼2.65.(i) Calculation of the Rius 2020,  is 1(ii) Calculation of the Fourier transform of |(iii) Calculation of ipp density modification procedure. Once completed, the modified η is Fourier-transformed to provide the new (iv) Calculation of the Fourier transform of η. Peakness in η is enhanced by applying the 4.e.g. of XLENS_v1 . In order not to have to modify the Fourier refinement module of already existing DM programs, H reflections with X ≥ 0.7.Notice that the first three factors of the left-hand side of (17)5.peak/σ units, where ρpeak is the density at the peak centre and σ2 is the variance of ρ.Relevant experimental information about the data sets used in the test calculations is given in Table 2Rfree values (listed in Table 2ref) were compared with the median Rfree values of the PDB which are 0.24, 0.25, 0.26 and 0.28 for upper resolution limits corresponding to the intervals 1.95–2.15, 2.15–2.35, 2.35–2.40 and ∼2.90 Å . The data set was measured at ID29 (ESRF) at the Zn absorption edge (λ = 1.282 Å) . Next, it was tested with more challenging cases. To simplify the discussion, the test compounds are divided into three groups.A preliminary test was the substructure solution of the proenzyme of proabylysin  and because the substitution of S by Se in the methionine amino acids is normally complete. The data sets of the three tested SeMet derivatives correspond to:Compared with other SAD situations, Se-SAD is particularly favourable due to the large AS strength of Se  XALOC  . There are 12 Se positions, two macromolecules and 509 water molecules in the a.u., amounting to 8119 atoms. Application of SAD-SMAR yields the positions of the 12 Se atoms (B2) with r.m.s.d. = 0.24 Å compared with the deposited refined model  . There are 18 Se, one Zn, two macromolecules and 104 water molecules in the a.u., amounting to 10 942 atoms. Application of SAD-SMAR supplies the positions of the 18 Se atoms (B2) with r.m.s.d. = 0.35 Å. Regarding the Zn ion, it shows up in the Fourier map 1.06 Å apart from the deposited refined position. Its strength is similar to that of the two Se atoms with higher B values.5lac: a = 94.144, b = 111.353, c = 58.191 Å, P21212. A 3C-like protease of Cavalli virus collected at BL 14.2  . There are 12 Se positions (one of them split in the refinement), one macromolecule and 303 water molecules in the a.u., amounting to 4875 atoms. Application of SAD-SMAR yields the positions of the 12 Se atoms (B2) with r.m.s.d. = 0.18 Å compared with the deposited model.5.2.Kα radiation) where the anomalous signal for I is large (−). The fifth case corresponds to crystals soaked in a Cd2+-containing solution.The first four cases of this subsection are native crystals soaked in a solution containing iodide ions and with their diffraction data being collected in-house on rotating anodes . According to the deposited data, there are 26 sites occupied by a total of 13.3 I1−, one macromolecule and 95 water molecules in the a.u. (1719 atoms). Application of SAD-SMAR yields 15 sites (B2) containing 9.74 I1− which show a good agreement with the deposited data (r.m.s.d. = 0.43 Å) as shown in Fig. 23k9g: a = 55.81, c = 200.90 Å, P4312. A plasmid partition protein . According to the structure refinement deposited in the PDB, there are 12 I1− sites, one macromolecule and 91 water molecules in the a.u. (1858 atoms) with 6.6 I1− in the 12 sites. Application of SAD-SMAR yields nine coincident I1− sites (B2) (r.m.s.d. = 0.35 Å) which justify a total of 5.3 I1−, i.e. 81% of the refined I1−content. By normalizing the sum of the heights of the nine strongest Fourier peaks to 5.3, the respective found and deposited site occupancies  are I1: 0.91, 0.99; I2: 0.91, 1.00*; I3: 0.60, 0.61; I4: 0.50, 0.53; I5: 0.59, 0.38; I6: 0.54, 0.38; I7: 0.60, 0.53; I10: 0.32, 0.36; I12: 0.33, 0.29 (* truncated to 1.00).3km3: a = 84.66, c = 140.74 Å, R3(H). A deoxycytidine triphosphate deaminase . The refinement in the PDB includes 16 I1− sites, two macromolecules and 516 water molecules in the a.u. (10 752 atoms) with 10.9 I1− in the 16 sites. SAD-SMAR gives 13 coincident I1− sites (B2) (r.m.s.d. = 0.43 Å) which justify the 87% of the refined I1−content. 3men: a = 45.70, b = 162.12, c = 173.07 Å, P212121. An acetylpolyamine aminohydrolase . According to the deposited data, there are 35 sites occupied by ∼23.1 I1−, four macromolecules and 516 water molecules in the a.u. (10 825 atoms). Application of SAD-SMAR yields 33 coincident I1− sites (B2) (r.m.s.d. = 0.24 Å) which justify ∼92% of the refined I1−content. The r.m.s.d. between the 33 found and corresponding deposited site occupancies is 0.172.2g4h: a = 182.16 Å, F432. A Cd-containing apoferritin measured at BL X12  . Anomalous signal for Cd2+ at λ = 2.00 Å is large (−). According to the deposited refinement, the a.u. contains five Cd2+ sites (with occupancies > 0.10), two Cl1− sites, 101 water molecules and one apoferritin subunit (a macromolecule with 1374 atoms). Apoferritin is made up of 24 such protein subunits which assemble to form a roughly spherical hollow shell, with an external diameter of ∼120 Å and an internal diameter of ∼80 Å  with the found positions and occupancies close to the deposited values (r.m.s.d. between corresponding sites is 0.32 Å). The respective found and deposited occupancies  are Cd1: 0.50, 0.50; Cd2: 0.25, 0.25; Cd3: 0.14, 0.20; Cd4: 0.20, 0.18; Cd5: 0.14, 0.16). The Cd1 sites are located pairwise (∼8 Å separation) at the 12 vertices of a cubo-octahedron centred at  (with opposite vertices separated by ∼129 Å), i.e. close to the external diameter of the hollow shell. The same applies for Cd2 but with a somewhat longer intra-pair distance (∼13 Å) and a separation between opposite vertices of ∼75 Å which roughly corresponds to the internal diameter of the hollow shell.5.3.Pyrococcus furiosus, were collected at BL X06DA at the Swiss Light Source .The data sets of Pf1117 and Pf0907, two hypothetical proteins from 4tno: a = 47.21, c = 82.28 Å; P41212. According to the deposited data, its a.u. contains one macromolecule, three methionine S atoms and two Cl1− . Application of SAD-SMAR yields the two Cl1− and two S atoms (B2). The third (more disordered) S atom could not be located. The r.m.s.d. between found and deposited positions is 0.48 Å.4pgo: a = 88.50, c = 73.12 Å; P6522. The deposited data indicate that besides the macromolecule and water molecules, there are two methionine S atoms and two Cl1− in the a.u. (∼689 atoms). Application of SAD-SMAR leads to the same AS model (B2) with r.m.s.d. = 0.56 Å.et al., 2006The third and last example is the PB1 domain of the human scaffold protein NBR1  . According to the deposited refinement, the a.u. contains, besides the macromolecule and the refined water molecules, four methionine S atoms  . Application of SAD-SMAR shows the four expected S atoms (B2), three of them as the three strongest Fourier peaks with a r.m.s.d. of only 0.18 Å compared with the deposited model. The fifth-ranked Fourier peak corresponds to the fourth S atom  and is shifted by 1.1 Å from the deposited position. The fourth-ranked Fourier peak could not be assigned (perhaps corresponding to some missing Cl1−).6.SMAR) between 2.50 and 3.3 Å; (ii) with average Bijvoet ratios of 0.065 (for SeMet derivatives), 0.014 (for S-SAD phasing) and 0.041 ; (iii) with D|) = 4 and DFCUT = ∼0.4. The introduction of DFCUT ensures the suppression of the less reliable |D|’s while keeping enough observations for a satisfactory DM run. It can be clearly seen that the corresponding CCh values are close to 0.88 for converging trials . Since for non-converging trials CCh values are normally smaller by 0.02–0.03 , identification of the correct trials should not be a problem. Notable is how quickly convergence is reached, especially for SeMet derivatives and for soaked native crystals. For native crystals with only S and/or Cl as AS, the test results clearly indicate that SAD-SMAR can be successfully applied to them. In the three test structures, the S atoms belong to methionine amino acids and no disulfide bridges are present. Since SAD-SMAR only considers the lattice symmetry operations, it processes the initial phase estimates derived from the randomly shifted M′ modulus function quite efficiently  with upper resolution limits  and the RCC values are 2 or 3 units larger . The comparison of the number of converging trials (n.c.t.) for both series of calculations indicates that DFCUT = ∼0.4 gives significantly higher n.c.t. values only for 2g4s and 4tno . This is surely related to their higher Rfree values  when compared with the median Rfree value of the PDB (0.265).To evaluate the influence of the DFCUT value in the phase refinement results, the test calculations were repeated with DFCUT = 0.0 and the results included in Table 3e.g. when the resolution of the data is not enough to resolve the AS peaks in the Fourier map. Fortunately, due to the large separation among anomalous scatterers, this limitation is generally not a problem. However, at intermediate resolutions (>2.0 Å), the presence of disulfide bridges in proteins, e.g. between cysteine residues, represents a limitation of the otherwise highly effective ipp procedure . This problem has already been addressed in SHELXD (Usón & Sheldrick, 2018ipp philosophy to the treatment of disulfide bridges would considerably expand the scope of SAD-SMAR in S-SAD phasing.One characteristic of SAD-SMAR is the delivery of almost complete models when it converges. Most probable causes of non-convergence are, besides the poor quality of the experimental data, some functional limitations of the model description, Click here for additional data file.10.1107/S2053273322008622/ae5119sup1.zipOutput of SAD_SMAR solutions, and comparison of SAD_SMAR efficiency for DFCUT=0.4 and DFCUT=0.000. DOI:


RUNX2. Disease-specific induced pluripotent stem cells (iPSCs) have emerged as a useful resource to further study human hereditary diseases such as CCD. In this study, we identified a novel CCD-specific RUNX2 mutation and established iPSCs with this mutation. Biopsies were obtained from familial CCD patients and mutation analyses were performed through Sanger sequencing and next generation sequencing. CCD-specific human iPSCs (CCD-hiPSCs) were established and maintained under completely defined serum, feeder, and integration-free condition using a non-integrating replication-defective Sendai virus vector. We identified the novel mutation RUNX2_c.371C>G and successfully established CCD-hiPSCs. The CCD-hiPSCs inherited the same mutation, possessed pluripotency, and showed the ability to differentiate the three germ layers. We concluded that RUNX2_c.371C>G was likely pathogenic because our results, derived from next generation sequencing, are supported by actual clinical evidence, familial tracing, and genetic data. Thus, we concluded that hiPSCs with a novel CCD-specific RUNX2 mutation are viable as a resource for future studies on CCD.Cleidocranial dysplasia (CCD) is an autosomal dominant hereditary disease associated with the gene  Cleidocranial dysplasia (CCD MIM 119600]) was first described as cleidocranial dysostosis by Marie and Sainton [19600) warunt-related gene 2 (RUNX2)  [hedgehog, FGF, WNT, and PTCH signaling genes [RUNX2 have been identified, including deletion, missense, nonsense, and frameshift mutations [RUNX2_c.371C>G in familial CCD patients and established CCD-specific hiPSCs bearing this novel RUNX2 mutation.The gene associated with CCD is d Cbfa1) –7, whichng genes . In chonng genes –12. The ng genes , 14. A nng genes . Both thng genes . In CCD utations , 18. Mosutations . The misutations –21. The utations . With thutations . Exampleutations . In thisThe patient (F4) Fig. A, an 8-yRUNX2 mutations, Sanger sequencing was performed with the primers we described previously [The patient’s genomic DNA was isolated from blood samples, dental pulp cells (DPCs) and CCD-DPC-hiPSCs with a QIAamp® DNA mini kit  according to the manufacturer’s protocol. To identify eviously . The PCRRUNX2 in this CCD family, targeted resequencing was performed with a MiSeq using a TruSight one panel , which is designed to comprehensively cover more than 4800 genes involved in diseases. Sequencing was done according to the manufacturer’s instructions using DNA derived from blood samples and dental pulp samples from F3 and F5. Data analysis was performed using the Illumina VariantStudio 3.0.To further search for pathogenic mutations in genes other than 2. Then, DPCs at a density of 1 × 105 cells were infected with SeVdp (KOSM) [4 cells/well onto a fibronectin (2 μg/cm2) -coated 6-well plate with serum-free hESF9 medium [2 as reported previously [Isolation of DPCs and induction of DPC-hiPSCs were performed as described previously . Brieflyp (KOSM) , which d9 medium –30, and eviously .OCT, NANOG, SOX2, and REX1) was performed using OnePCR™ . Next, to determine the in vitro differentiation capacity of the CCD-DPC-hiPSCs, we performed an embryoid body (EB) assay. Differentiated cells were fixed, stained with antibodies , and visualized under a Zeiss inverted LSM 700 confocal microscope .The hiPSCs were characterized as described previously , 32. Bri5 cells per well were in seeded in a low attachment U bottom prime surface 96-well plate in hESF6 without FGF2 and heparin and cultured for 7 days to make EBs. Then, 5–7 EBs were transferred to a gelatin-coated 35 mm dish. After approximately 7 days, mesenchymal stem-like cells had grown out from the EBs and spread to confluence in the dish. To remove the EB mass, the detached cells were filtered through a 70 µl filter. After several passages through the filter using hESF9 with FGF2 and heparin, the cells were analyzed with FACS  using anti-MSC marker  antibodies (Miltenyi Biotec) on 4th week. The induced MSCs were seeded on a fibronectin-coated 60 mm dish at a concentration of 1.0 × 106 cells per dish with hESF9. Chondrogenic medium consisted of hESF6f as a base medium supplemented with Wnt3A (10 ng/ml)  and activin (10 ng/ml) (R&D) for 4.5–4.7 weeks and then BMP2 (50 μg/ml) (R&D), TGFβ1 (10 ng/ml) (R&D), GDF5 (10 ng/ml) , and FGF2 (10 ng/ml) (R&D) for 4.7–10 weeks. On the 6th week, the cells were detached in a sheet using a p-1000 pipet tip, transferred to a 15 ml tube, centrifuged at 200×g for 5 min , and kept in an incubator at 37 °C in a humid atmosphere of 95% air/5% CO2. The tube was centrifuged at 7×g for 5 min each day over 6–10 weeks. This protocol was a modified version of the process for chondrogenic differentiation of hiPSCs reported by Yamashita et al. [The hiPSCs were detached with TrypLE/ EDTA, and 1 × 10a et al. . At 10thRUNX2_c.371C>G was detected in individuals F3, F4, and F5 . Though SIL1_c.617T>A was predicted to be ‘disease-causing’ in silico, the detected mutation was heterozygous, indicating non-pathogenicity. SOX9, the Sox family of transcriptional regulators, is known to function during chondrocyte differentiation, and its deficiency leads to skeletal malformation [SIL1_c.617T>A and SOX9_c.724A>C mutations from the group of mutations considered pathogenic. The base substitution ‘c.371C>G’ in RUNX2, which was predicted to be ‘disease-causing’ by Mutation Taster, ‘probably damaged’ by PolyPhen-2, and ‘deleterious’ by the SIFT software, indicates a substitution of serine to tryptophan at codon 124, p.S124W, which is in the RHD. Based on this information, we predicted that RUNX2_c.371C>G_p.S124W was a ‘likely pathogenic’ mutation for CCD. However, RUNX2_c.371C>G_p.S124W was not found in Togo Var or Exome Variant Server, whereas in dbSNP and Clinvar, this mutation was submitted as NM_001024630.4(RUNX2):c.371C>G (p.Ser124Trp) in January 2021 and classified as a variant of uncertain significance (VUS). However, as far as we have investigated, clinical case of CCD with this mutation has not been reported anywhere.Using Sanger sequencing, ormation . HoweverOCT, NANOG, SOX2, and REX1 in PCR analyses (Fig. CCD-derived disease-specific hiPSCs inherited the RUNX2 mutation c.371C>G Fig. A. The ceses Fig. B. In vitses Fig. C. TogethThe algorithms of SIFT, PolyPhen-2, and Align-GVGD, etc. were recently developed to predict the effects of missense changes on protein structures . AccordiThe CCD family reported in this study exhibited the common phenotypes of open suture, hypoplastic clavicles, and supernumerary teeth. Although the patients showed no delay in fontanelle closure, in general, our results matched those of a previous study . Short sPatients F4 and F5 had 8 and 10 supernumerary teeth, respectively. Previous studies have reported that in patients with CCD, the number of supernumerary teeth ranged from 0 to 21, and that these teeth were frequently found as maxillary incisors and mandibular premolars , 38. UndRUNX2 mutations using CCD-derived iPS cell lines may contribute to the development of regenerative therapies for CCD.In conclusion, we report a novel mutation, RUNX2_c.371C>G_p.S124W, in Japanese familial CCD. Based on the clinical evidence and preliminary data mining using online tools and software, RUNX2_c.371C>G_p.S124W is a ‘likely pathogenic’ mutation for CCD. Based on previous findings and those of the current study, analyses of


We evaluated the effects of 2 direct-fed microbial (DFM) supplements containing 4 native rumen microorganisms on the production of dairy cows. Ninety Holstein cows (43% primiparous) were fed a common diet. Mean days in milk, milk yield, and body weight at the beginning of the study (mean ± standard deviation) were 92 ± 23 d, 45 ± 10 kg/d, and 659 ± 86 kg, respectively. After 14 d, they were blocked by parity, days in milk, and energy-corrected milk (ECM) per unit of metabolic body weight. Within block, cows were randomly assigned to treatments, which were top-dressed daily for the next 112 d. Treatments were 150 g of ground corn mixed with (1) no live DFM (CON), (2) 5 g of a live DFM , and (3) 5 g of a live DFM . G2 and G2P were products of Native Microbials Inc.  and contained the same organisms but in different concentrations. Supplementation with DFM did not alter yield of total milk, protein, or fat, but slightly decreased body weight gain and body condition score gain with no difference between G2 and G2P. The DFM tended to decrease dry matter intake (DMI) and tended to improve ECM/DMI. The DFM did not alter digestibility of fiber, starch, or protein and did not alter concentrations of glucose or nonesterified fatty acids, but tended to decrease concentration of insulin in plasma. Direct-fed microbials decreased somatic cell counts in milk with no difference between G2 and G2P. In conclusion, supplementation with native DFM had little impact on animal production and efficiency. DFM) are feed additives commonly used in dairy production to promote milk production, efficiency, and health. These products, composed of viable yeast and bacteria, are fed to manipulate the rumen and lower gut microbiome  have enhanced starch and fiber digestion in vitro  in a randomized complete block design. Mean DIM, milk yield, and BW for all cows at the beginning of the study (mean ± SD) were 92 ± 23 d, 45 ± 10 kg/d, and 659 ± 86 kg, respectively. Within cohort, cows were blocked by parity, DIM, and ECM/BWAll experimental procedures were approved by the Michigan State University Institutional Animal Care and Use Committee.CON), (2) 5 g of a live DFM (G2) containing Clostridium beijerinckii at 1.0 × 107 cfu; Pichia kudriavzevii at 1.0 × 108 cfu; Ruminococcus bovis at 1.0 × 108 cfu; and Butyrivibrio fibrisolvens at 1.0 × 108 cfu , or (3) 5 g of a live DFM (G2P) that was similar to G2 but contained more C. beijerinckii (7.5 × 107 cfu) and P. kudriavzevii . Treatments were mixed into the top 15 cm of each cow's feed before she had access to it. Treatments were obtained from the manufacturer every 3 mo, stored at 2°C, and mixed with corn daily before feeding.Cows were fed a common diet for a 14-d preliminary period, and then treatments were top-dressed on the common diet for 112 d. Daily treatments were 150 g of ground corn mixed with (1) no DFM  or 800 h (cohort 2), and orts were recorded before feeding to adjust the amount of feed offered. Water was available ad libitum in each stall. Body weight was measured for each cow 3 d per week. Body condition was scored by 3 trained investigators on a 5-point scale in 0.25 increments  at −14, Daily milk yield was automatically recorded at each milking. Milk samples were collected for 6 consecutive milkings per week for component analysis. Samples were stored with preservative  at 4°C until analysis. Individual milk samples were analyzed by CentralStar Cooperative Inc. for fat, true protein, lactose, MUN, and SCC concentrations by mid-infrared spectroscopy  were collected once weekly and stored at −20°C until composited by month and dried; these values were used for calculating final diet composition . Appareng for 15 min at 4°C and stored at −20°C until composited by cow and analyzed. Plasma nonesterified fatty acid (NEFA), glucose, and insulin concentrations were analyzed using commercially available kits according to the manufacturer's instructions . Absorbance was measured with a micro-plate reader .From each cow in the digestibility study, blood (~15 mL) was sampled at each fecal collection and on 93 ± 1 d at −1, +2, and +6 h after feeding into EDTA- and sodium fluoride-coated tubes. Plasma was harvested after centrifugation at 2,000 × ijkl = dependent variable, Cov = fixed effect of 2-week covariate period, μ = overall mean, Ci = fixed effect of cohort (i = 1 to 2), Pj = fixed effect of parity (j = 1 to 2), Ci′Pj = interaction of cohort and parity, Bk(Ci′Pj) = random effect of block within cohort and parity, Tl = fixed effect of treatment (l = 1 to 3), Tl′Ci = interaction of treatment and cohort, Tl′Pj = interaction of treatment and parity, Tl′Ci′Pj = interaction of treatment, cohort, and parity, and eijkl = residual error. Week on treatment was originally included in the model but deleted because it was not significant. Body weight gain/d and BCS gain/28 d were analyzed using the same model but excluding the covariate variable. Normality of results was tested using box plots, normal probability, and homogeneity of variances. Somatic cell count displayed nonnormality so was log-transformed for analysis. Main effects were declared significant at P ≤ 0.05 and tendencies at 0.05 < P ≤ 0.15. All data were expressed as LSM and SEM, unless otherwise specified. Preplanned contrasts were CON versus DFM and G2 versus G2P.All data were analyzed using the mixed model of SAS  according to the following model:P > 0.4). Treatment tended to decrease milk fat concentration slightly in cohort 2 but not in cohort 1 (P = 0.07 for T′C interaction). Average SCC of CON was 20,700 cells/mL; DFM decreased SCC (P = 0.05), but G2 and G2P were not different (P = 0.28). Average BW was 690 kg and BCS was 3.2. Treatments altered BW gain  and BCS gain . The effect for BW gain was most pronounced in cohort 1 (P = 0.02 for T′C interaction) where animals fed the DFM treatment on average gained 0.19 kg/d less than CON in cohort 1 but 0.07 kg/d less than CON in cohort 2. No differences between G2 and G2P were detected for gain in BW or BCS (both P > 0.7).On average, cows produced 44 kg of milk daily with 3.9% fat, 3.2% true protein, 4.9% lactose, and 13 mg/mL of MUN . Yields P = 0.08) with the decrease tending to be greater in G2P than G2 (P = 0.14). The DFM tended to increase ECM/DMI (P = 0.06) from 1.54 to 1.58, and this difference was due to cohort 1; ECM/DMI was 1.55 for CON and 1.61 for DFM in cohort 1 but 1.54 for both CON and DFM in cohort 2 (P = 0.14 for T′C interaction). No difference was detected between G2 and G2P. The DFM did not improve captured energy over feed energy (P > 0.5). Apparent total-tract digestibility of OM tended . Apparent total-tract digestibilities of starch, NDF, and CP were not altered by treatment .Average DMI of CON cows was 29.6 kg/d, and DFM tended to decrease DMI by 0.6 kg/d , but the interaction of cohort and treatment also tended to be different (P = 0.07) with a 24% decrease in cohort 1 but a 3% increase in cohort 2. No difference in plasma insulin was observed for G2 compared with G2P (P = 0.6). Compared with CON, DFM treatments did not alter plasma NEFA concentration (P = 0.4) but G2P had greater NEFA concentration than G2 (P = 0.03). Plasma glucose concentration was not affected by treatment (P = 0.3). There was no significant interaction of cohort and treatment for plasma glucose and NEFA concentrations (both P > 0.2). All 3 blood measures were affected by time relative to feeding . For times −1, +2, and +6 h relative to feeding, plasma insulin averaged 0.44, 1.10, and 0.87 μg/L, plasma glucose was 58, 51, and 54 mg/dL, and plasma NEFA was 84, 70, and 73 μM, respectively. The DFM treatments tended (P = 0.12) to decrease NEFA after feeding relative to controls but had no effect on feeding responses for insulin and glucose.Average plasma glucose, NEFA, and insulin concentrations were 55 mg/dL, 88 μectively . TreatmeC. beijerinckii and P. kudriavzevii, and also saw no change in production.Based on the tendency for lower OM digestibility and DMI of cows fed the native DFM, we suggest the native DFM altered rumen fermentation to decrease the postfeeding spikes in propionate and caused the trend for less plasma insulin. Lower concentrations of plasma insulin may have resulted in partitioning available energy toward milk production instead of body tissue and thus reducing gains with no drop in milk yield. Additionally, DFM did not alter plasma NEFA concentrations indicating fat mobilization was not altered to sustain production. The lack of effect on milk production and composition in this study is similar to a study by R. bovis and C. beijerinckii were expected to enhance starch digestion  and concentrations of plasma metabolites  were unaffected.


Escherichia coli cells, but some of these methods were inconvenient, using heterologous systems, or relied on engineered E. coli strains.Natural life systems can be significantly modified at the genomic scale by human intervention, demonstrating the great innovation capacity of genome engineering. Large epi-chromosomal DNA structures were established in E. coli has a single circular chromosome. In this work, a novel method was developed to split the original chromosome of wild-type E. coli. With this method, novel E. coli strains containing two chromosomes of 0.10 Mb and 4.54 Mb, and 2.28 Mb and 2.36 Mb were created respectively, designated as E. coli0.10/4.54 and E. coli2.28/2.36. The new chromosomal arrangement was proved by PCR amplification of joint regions as well as a combination of Nanopore and Illumina sequencing analysis. While E. coli0.10/4.54 was quite stable, the two chromosomes of E. coli2.28/2.36 population recombined into a new chromosome (Chr.4.64MMut), via recombination. Both engineered strains grew slightly slower than the wild-type, and their cell shapes were obviously elongated.The wild-type model bacterium E. coli strains with no heterologous genetic parts. This technique might be applied to other prokaryotes for synthetic biology studies and applications in the future.Finally, we successfully developed a simple CRISPR-based genome engineering technique for the construction of multi-chromosomal The online version contains supplementary material available at 10.1186/s12934-022-01957-4. Saccharomyces cerevisiae by assembling DNA fragments, and then installed those synthetic genomes into recipient Mycoplasma cells [Saccharomyces cerevisiae into a single functional chromosome [Genome engineering is defined as the design and modification of genomic DNA sequences for a specific purpose. In 2010, U.S. scientist J. Craig Venter constructed circular chromosomes in romosome . This woS. cerevisiae, we aimed to design a simple technique to create an artificial multi-chromosomal prokaryotic organism, such as E. coli. There were reports that large epi-chromosomal DNA structures were established in E. coli cells. The group of G. Church employed the λ attB-attP recombination method to construct a large F-based BAC vector [E. coli strain, Yoneji et al. split the 3 Mb chromosome into three 1 Mb chromosomes [Vibrio tubiashii secondary chromosome. Although great achievements have been made in synthetic biology, some of these methods were inconvenient, and heterologous systems were employed, such as the F-based par locus and the BAC vector. Furthermore, since an engineered E. coli strain was used for the chromosome splitting, it remained unknown if wild-type strains could be manipulated using these methods.The technological basis of genome engineering is the construction and implementation of novel genetic systems and remodeling of natural biological systems. In contrast to the single-chromosome C vector . Startinomosomes . The firE. coli strain MG1655, we aimed to develop a CRISPR-based genome engineering technique for the design and modification of genomes as desired. With this technique, we constructed a multi-chromosomal E. coli strain with no heterologous sequences.In this work, starting from the wild-type E. coli chromosome are the origin of replication and 10 replication terminators [E. coli chromosome is initiated from a single origin. Two replication forks, driven by the replication machinery, travel in opposite directions around the circular chromosome and terminate in a region opposite the origin. The DNA replication terminators, designated as Ter, each contain a consensus element with a length of 23 bp. Ter sites are recognized by Tus (terminus utilization substance) protein, forming a protein-DNA complex, which halts the passage of the replication fork in only one direction and permits passage by the other replication fork. A group of Ter sites with the same polarity are distributed in one half of the terminus region, and the other group, with the opposite polarity, is located in in the other half. Our first goal was to separate a 100 k bp DNA fragment out of the chromosome to form a second chromosome without perturbation of the Ter loci using a CRISPR-based genome engineering technique.The key components maintaining an minators . The bidE. coli chromosome. The separated second origin of replication was used to avoid the presence of two origins on a single chromosome. In the second step, two DNA double strand breaks were introduced by the Cas9/gRNA complex on the two inserted loci, which induced two intramolecular recombination events of the chromosome. As designed, the two halves of the replication origin combined into a complete origin and formed a circular DNA molecule containing all DNA sequences cleaved from the original chromosome, while the rest of the chromosome recombined into a second circular DNA molecule containing the original replication origin. Thus, two new chromosomes derived from the original one were formed in a single E. coli cell.For this purpose, we designed a CRISPR-based chromosomal-separation technique Fig. . First, 1/R1  and L2/R2 , on the E. coli chromosome, located 0.10 Mb away from each other. After Cas9/gRNA-induced intramolecular recombination, the two halves of the replication origin recombined into a complete one, forming Chr.0.10 M containing the 0.10 Mb DNA sequence between them. At the same time, Chr.4.54 M was constructed by a recombination event between the L1_up and L2_low homologous arms.To construct a second chromosome of 0.10 Mb, two halves of the replication origin were inserted into two loci, LE. coli containing two chromosomes of 0.10 Mb and 4.54 Mb (E. coli0.10/4.54 strain), a single colony was picked for culture to ensure the purity of the population. The harvested cells were used as the template of the identification PCR. Four primer pairs were designed to amplify the key regions spanning the L1/R1, L2/R2, L1/R2 and L2/R1 sequences, respectively  and f2/r2 (2956 bp). After the CRISPR-induced recombination event, the E. coli0.10/4.54 strain was created. To confirm the successful construction, PCR was performed with another two pairs of primers as illustrated in Fig. 1/r2 (1093 bp) and f2/r1 (3015 bp), in addition, the colony PCR bands that met the expected size were confirmed by DNA sequencing, which proved that a chromosome of 4.54 Mb and a second chromosome of 0.10 Mb were successfully split from the original chromosome of E. coliC1/C2.To identify the constructed multi-chromosomal E. coli0.10/4.54 strain  was separated in two halves and inserted into the chromosome along with the split E. coli origin of replication. Thus, after CRISPR-induced chromosomal DNA recombination, a complete ampr gene was pieced together from the halves in addition to a second origin of replication that was also newly formed  and f4/r4 (3581 bp).To obtain a strain bearing two large chromosomes, we improved the genome engineering technique by implementing a selective pressure for stains carrying the newly created chromosomes. An ampicillin resistance gene  and f4/r3 (3479 bp). The PCR identification experiment proved that a chromosome of 2.28 Mb and a second chromosome of 2.36 Mb were successfully split from the E. coliC3/C4 chromosome, and the resulting strain was designated as E. coli2.28/2.36.After the recombination event, an E. coli2.28/2.36 .Similarly, de novo genome sequencing using both Nanopore and Illumina methods was carried out to further analyze the genomic DNA of Mut was assembled at the same time. Careful analysis revealed that Chr.4.64MMut was reassembled from Chr.2.28 M and Chr.2.36 M by a homologous recombination based on a sequence of 1195 bp (Marked as H), which was located at insH-8 on Chr.2.28 M (L5/R5) and insH-9 on Chr.2.36 M (L6/R6) and appeared 10 times in total on the Chr.4.64MMut chromosome  and Chr.4.64MMut (①-③`-④`-②).Unexpectedly, a new Chr.4.64ME. coli strain. To study the stability of the chromosomal organization, an E. coli0.10/4.54 of 0 generations that had just been identified correctly was transferred in batch culture for more than 100 generations without antibiotics , by a homologous recombination based on H sequence, which appeared 10 times in total on the Chr.4.64MMut chromosome. Deletion of all of them was not practical. Yoneji et al. obtained three 1 Mb genomes in 2021 from a very special E. coli strain, the 3 Mb chromosome of DGF-298 W, the recBCD genes and all insertion sequence (IS) of which were deleted using a stepwise genome reduction approach. DGF-298 W strain showed no auxotrophy, with fewer genes and better cell yield and better growth fitness in a rich medium than the wild type K-12 strain [E. coli MG1655 used in this study. The group of G. Church employed the λ attB-attP recombination method to construct a large F-based BAC vector [E. coli strain MG1655, we develop a CRISPR-based genome engineering technique for the design and modification of genomes as desired. With this technique, we constructed a multi-chromosomal E. coli strain with no heterologous sequences. The CRISPR-based chromosomal-separation technique might be further developed by combining it with other methods, such as the rapid giant DNA production in Bacillus subtilis [Streptomyces chassis [E. coli genome into two defined pairs of synthetic chromosomes provides common intermediates for large-scale genome manipulations such as inversion and translocation. Precise, rapid, large-scale genome engineering operations are useful tools for creating diverse synthetic genomes.This work provides a set of simple and rapid engineering techniques for creating multi-chromosomal subtilis , the mul chassis . ImportaE. coli has a single circular chromosome. In this work, we developed a CRISPR-based chromosomal-separation technique to create a multi-chromosomal E. coli. Using this method, we first created the multi-chromosomal E. coli0.10/4.54 strain containing two chromosomes of 0.10 Mb and 4.54 Mb, respectively, which was proved by both colony PCR and a third-generation genome sequencing method. The chromosomal organization of E. coli0.10/4.54 was highly stable, and no changes were observed after more than 100 generations of culture without antibiotics. Using the same approach, the E. coli2.28/2.36 strain containing two more similarly sized chromosomes, Chr.2.28 M and Chr.2.36 M, was created. However, the chromosomal organization of E. coli2.28/2.36 was not stable, and the two large chromosomes were found to recombine into a new chromosome (Chr.4.64Mut) after about 70 generations of culture completely, that we couldn’t get a stable E. coli2.28/2.36 strain, even if the tested sample was grown from a single cell or a single colony. We found that both genome-engineered strains grew slightly slower than the wild type, and their cell shapes were obviously elongated to 3, 4, or 5 μm, and in some cases even longer, as observed by scanning electron microscopy.The wild-type model bacterium Escherichia coli DH5α was used as a cloning host. Wild-type E. coli MG1655 was used in the genome duplication experiments. Strains were grown at 30 °C in Luria–Bertani medium  tryptone, 0.5% (w/v) yeast extract, and 1% (w/v) NaCl). Kanamycin (50 mg/L), chloramphenicol (30 mg/L), ampicillin (100 mg/L), spectinomycin (100 mg/mL) and apramycin (50 mg/L) were added to the medium when appropriate. One percent (w/v) glucose and 2 g/L l-arabinose were added to the culture for the repression and induction of Cas9 expression, respectively.Escherichia coli MG1655 cells with or without plasmids were grown in 50 ml of LB medium supplemented with the appropriate antibiotics at 30 °C to OD600 = 0.6, and then made electro competent by concentrating 100-fold and washing three times with 10% ice-cold glycerol. Then, 50 ng of plasmid DNA or 800 ng of the editing cassette was used for electroporation. The shocked cells were resuspended in 1 mL of LB, incubated for 2 h at 30 °C, and spread on LB agar plates with the appropriate antibiotics.1/R1) and pgRNA(L2/R2) used for the creation of the E. coli0.10/4.54 strain were constructed using Golden Gate assembly [1/R1) and pgRNA(L2/R2) were constructed for guiding CRISPR/Cas9 to two target loci, L1/R1  and L2/R2 , on the chromosome of E. coli MG1655. The backbones of plasmids pgRNA(L1/R1) and pgRNA(L2/R2) were PCR amplified from pACYC184-M [poxB300 and assembled using the Golden Gate method. For the creation of the E. coli2.28/2.36 strain, the inducible gRNA plasmids pgRNA(L3/R3) and pgRNA(L4/R4) were constructed using the same method for guiding CRISPR/Cas9 to two target loci, L3/R3  and L4/R4 , on the chromosome of E. coli MG1655.The plasmids pgRNA from the left part of the paaY gene on the chromosome, a CRISPR/Cas9 recognition region (N20PAM), the right half of the replication origin (ori_R), and a right homologous arm (R1) from the right part of the paaY gene on the chromosome. Five modularized parts were also prepared with same method for assembly of the editing cassette 2, which included a left homologous arm (L2) from the left part of the dosP gene on the genome, a complete chloramphenicol resistance gene (cat), the left half of the replication origin (ori_L), a CRISPR/Cas9 recognition region (N20PAM), and a right homologous arm (R2) from the right part of the dosP gene on the chromosome. Modularized parts of editing cassette 1 and editing cassette 2 were modified and optimized for construction of editing cassette 3 (C3) and editing cassette 4 (C4) for the creation of E. coli2.28/2.36. In the construction of editing cassette 3, ampr_L was added to the editing cassette 1, L1 was replaced with a left homologous arm (L3) from the left part of the bolA gene, and R1 was replaced with a right homologous arm (R3) from the right part of the bolA gene. At the same time, editing cassette 4 was constructed, in which ampr_R was added to the editing cassette 2, the cat gene was replaced with a complete bleomycin resistance gene (ble), L2 was replaced with a left homologous arm (L4) from the left part of the pheA gene, and R2 was replaced with a right homologous arm (R4) from the right part of the pheA gene.The editing cassette 1 (C1) and editing cassette 2 (C2) used for the creation of 1 and at the front of R2, which was used to cyclize chromosome Chr.4.54 M; two pairs at the end of ori_L and at the front of ori_R, which were used to reconstruct the entire replication origin and cyclize two chromosomes: Chr.0.10 M and Chr.2.28 M respectively; the last pairs at the end of ampr_L and at the front of ampr_R, which were used to reconstruct the entire ampicillin resistance gene and cyclize chromosome Chr.2.36 M. Four Golden Gate reactions were performed to assemble these parts into the corresponding editing cassettes. The L and R homologous arms of each editing cassette (about 500 bp each) were amplified from the genomic DNA of E. coli MG1655. The selection markers  with the CRISPR/Cas9 recognition region (N20PAM) were PCR-amplified from the plasmids pETDuet1 pPICZαA and pACYCDuet1 with the N20PAM sequence embedded in the reverse primer.There were four pairs of identical sequences of 40 bp. After CRISPR/Cas9 cleavage induced homologous recombination, one pair at the end of LDpnI before assembly. Primers for the construction of the plasmids and editing cassettes, as well as other primers are summarized in Additional file All the DNA templates were PCR-amplified using Phusion polymerase . PCR products were purified by preparative agarose gel electrophoresis using the AxyPrep DNA Gel Extraction Kit , and the template was digested with Escherichia coli MG1655 competent cells harboring pRedCas were prepared by inducing the expression of the CRISPR-Cas9 system and λ-RED proteins using l-arabinose. An aliquot comprising 50 μL of the competent cells was mixed with 50 ng of pgRNA(L1/R1) and 800 ng of editing cassette 1 DNA in a 2-mm Gene Pulser cuvette . After electroporation at 2.5 kV and immediate resuspension in 1 mL of ice-cold LB medium, the cells were incubated for 2 h at 30 °C, and then spread on LB agar plates with kanamycin and apramycin. For each editing experiment, ten transformants were identified by colony PCR with the f1/r1 primer pairs illustrated in Fig. 1/R1) plasmid with the apramycin resistance gene. A single colony that grew on kanamycin plates but did not grow with apramycin was selected for subsequent experiments. Subsequently, the HaploidC1/C2E. coli strain with chloramphenicol resistance was obtained by integrating editing cassette 2 into the E. coli chromosome in the same way as described above. Finally, the E. coliC1/C2 harboring pRedCas9 was then transformed with the pgRNA(N20PAM) plasmid through electroporation. Transformants were grown in LB medium at 30 °C with appropriate antibiotics for 2 h, after which 2 g/L l-arabinose was added to induce the expression of the CRISPR-Cas9 system and λ-RED proteins. After overnight culture, the cells were spread on LB agar plates with kanamycin, apramycin and chloramphenicol. To identify correctly edited clones, ten colonies were analyzed by colony PCR with three primer pairs designed to amplify the key regions as illustrated in Fig. E. coli0.10/4.54 was created. After finishing all genome modifications, all editing plasmids were eliminated by growing overnight at 42 °C.The genome engineering process is illustrated in Fig. E. coli2.28/2.36 and E. coli0.10/4.54 was slightly different. In the last step, a CRISPR/Cas9 system was expressed with gRNA(N20PAM) to induce two double-strand breaks (DSBs) at two N20PAM sequences inserted as part of the editing cassettes 3 and 4, which caused a DSBs-mediated intra-chromosomal recombination event between the ampr_L and ampr_R homologous arms. Thus, a new ampicillin resistance gene was generated in E. coli2.28/2.36 that was not present in E. coli0.10/4.54. To identity correctly edited clones, ten colonies were analyzed by colony PCR with three primer pairs designed to amplify the key regions as illustrated in Fig. E. coli2.28/2.36 was created. After finishing all genome modifications, all editing plasmids were eliminated by growing overnight at 42 °C.The construction of E. coli strains were sequenced using a Nanopore sequencing platform. Cells of E. coli0.10/4.54 and E. coli2.28/2.36 were harvested, and all further experimental procedures were carried out according to the standard protocol provided by Oxford Nanopore Technologies (ONT), including sample quality control, library construction, library quality control and library sequencing. In the assembly process, the genome was assembled using high-accuracy Illumina data (Q30 > 85%) and nanopore reads using Unicycler (0.4.8) and Tricycler (0.5.3) software to obtain high quality contigs, and finally Illumina reads were used for error correction in Pilon software.The whole genomes of the two engineering E. coli and wild-type E. coli were continuously transferred in batch culture at a 1‰ inoculation ratio without antibiotics, and cultured overnight for 16–18 h to OD600 = 5. A total of 11 rounds of inoculation were conducted, corresponding to approximately 100 generations. The culture of the first (0 generation), third (about 30 generations), fifth (about 50 generations), seventh (about 70 generations), ninth (about 90 generations) and last (about 100 generations) round of E.coli2.28/2.36 were spread on LB agar plates to investigate the speed of transition of genomic instability with the passage of generations, while the first and last round culture of wild-type E. coli and E.coli0.10/4.54 cultures were spread on LB agar plate to investigate the chromosomal stability. Thirty colonies from the each LB agar plate were examined by colony PCR, using the above mentioned sets of identification primers.Two engineered E. coli and wild-type E. coli were cultured without antibiotics. The growth rates during the exponential phase were evaluated according to the growth curves. The doubling time (DT) was calculated based on two continuous reading points in a growth curve, according to the equation, below:600 values at two continuous time points of tj and ti, which were at intervals of either 0.5 or 1 (h) in the present study. Every four to five continuous growth rates that exhibited the largest mean and the smallest standard deviation were averaged to calculate the exponential doubling time for the growth curve.Two engineered E. coli and two engineered E. coli in the exponential phase were harvested, and washed three times with phosphate buffered saline . The sample were fixed for 2 h in 2.5% glutaraldehyde and post-fixed for 1 h with 1% of osmium tetroxide. The samples were dehydrated with ethanol and dried in an Automated Critical Point Dryer (Leica EM CPD300). Then, the samples were coated with platinum and observed under a scanning microscope (Hitachi SU8010).For scanning electron microscopy (SEM), cells of the wild-type Additional file 1: Table S1. Plasmids and E. coli stains used in this study. Table S2. Primers used in this study. Table S3. The schematic and sequences of the editing cassette used in this study. Table S4. The sequences of clone PCR products used to identity the E. coli variants. Figure S1. Stability of the chromosomal organization of E. coli0.10/4.54 and E. coli2.28/2.36. (A) Colony PCR analysis of the E. coli0.10/4.54 cells after culturing for more than 100 generations using the primer pairs f1/r1, f2/r2, f1/r2 and f2/r1. (B) Colony PCR analysis of E. coli2.28/2.36 after culturing for more than 100 generations using the primer pairs f3/r3, f4/r4, f3/r4 and f4/r3. Figure S2. Stability of the chromosomal organization of E. coli2.28/2.36 (mix with E. coliMut). A total of 11 rounds of inoculation were conducted, corresponding to approximately 100 generations. The culture of the first (0 generation), third (about 30 generations), fifth (about 50 generations), seventh (about 70 generations), ninth (about 90 generations) and last (about 100 generations) round of E. coli2.28/2.36 (mix with E. coliMut) was spread on LB agar plates, 24 single colonies of each strain were selected and four pairs of primers f5/r5, f6/r6, f5/f6 and r5/r6 were used to investigate the speed of transition of genomic instability with the passage of generations.


The active ingredients in extracts of leaves of artichoke, Cynara scolymus L., were determined by HPLC analysis. In the 42-day experiment, rats were exposed to either sterile water, 4% DMSO, 100 mg artichoke leaf extract/kg body mass, 72 μg aflatoxin B1/kg body mass, or AFB1 plus artichoke leaf extract. Neurotoxicity of AFB1 was determined by an increase in profile of lipids, augmentation of plasmatic glucose and concentrations of insulin, oxidative stress, increased activities of cholinergic enzymes, and a decrease in activities of several antioxidant enzymes and pathological changes in brain tissue. Extracts of artichoke leaf significantly reduced adverse effects caused by AFB1, rescuing most of the parameters to values similar to unexposed controls, which demonstrated that adverse, neurotoxic effects caused by aflatoxin B1 could be significantly reduced by simultaneous dietary supplementation with artichoke leaf extract, which itself is not toxic.Attenuation of adverse effects of aflatoxin (AFB Alternaria, Fusarium, and Cladosporium, which reputedly gain access to seeds during plant development. The second class is described as “storage” fungi and includes species from the genera Penicillium and Aspergillus that proliferate during storage [Aspergillus flavus and Aspergillus parasiticus produce aflatoxins (AFs) that can infect crops before harvesting. Contamination of field crops with aflatoxins causes losses of agricultural commodities in many zones of the world [Mycotoxins are toxic fungal products that thrive in human food and animal feed, causing disease and death in humans and animals, when consumed in concentrations exceeding the limits , 2. Thes storage . Both Ashe world . Therefo1) is considered the most significant risk factor among mycotoxins and the most frequently found aflatoxin in contaminated foods and feed [1. Therefore, public health can be affected following dietary exposure to AFB1. On a worldwide basis, the safe limits of aflatoxin B1 range from 4 to less than 30 μg/kg for human consumption [1 may increase lipid peroxidation, which contributes to cytotoxic damage in many organs, especially the brain, leading to neurodegenerative diseases and weakening of this organ's function [1 in rat offspring can delay development of reflex responses, learning ability, motor activity, locomotor coordination, and exploratory behavior [1 has been reported to alter concentrations of neurotransmitters in brains of rats [1 was investigated in which diets were supplemented with an entirely natural, plant-based product, extracts of leaves of artichoke. The alternative therapy if effective would be cost-effective and result in fewer side effects.Aflatoxin B1  and free radicals and perform as a protective shield against oxidative damage to biological molecules, such as proteins, lipids, and DNA . Gebhard1 can be significantly reduced by simultaneous dietary supplementation with extracts of leaves of artichoke.Effects of artichoke leaf extract (ALE) after 42 days of exposure in male rats were investigated. General pathological characteristics and specific brain plasma markers were evaluated. Besides, lipid profile, antioxidant enzyme activities, and free radicals were analyzed in the brain to elucidate the potential neuroprotective effects of ALE. Furthermore, the histopathological architecture of the brain was evaluated. In this study, the following hypothesis was tested: adverse neurotoxic effects caused by aflatoxin B1 powder (C17H12O6) from Aspergillus flavus was purchased from Sigma-Aldrich, France . Commercial ALE  was produced by Western Pharmaceutical Industries, Cairo, Egypt, and used in this study. All reagents and chemicals were of analytical grade.AflatoxinBad libitum. Animals were maintained in a controlled atmosphere at a temperature of 25 ± 5°C and 50-70% humidity, and they were subjected to standard 12 h light and dark cycles.Twenty-five 12-week-old male albino rats were used for this research. The rats were obtained from the Medical Research Institute , Alexandria University, Egypt. They were housed in clean cages with hardwood bedding. They had access to feed and clean tap water μm). Water and 0.05% trifluoroacetic acid in acetonitrile were used at a flow rate 1 mL/min as mobile phases A and B, respectively. The gradient was programmed consecutively in a linear manner as follows: 0 min (82% A), 0–5 min (80% A), 5-8 min (60% A), 8-12 min (60% A), 12-15 min (82% A), 15-16 min (82% A), and 16-20 (82% A). The multiwavelength detector was monitored at 280 nm. The injection volume was 5 μL for each of the sample solutions. The column temperature was maintained at 40°C.HPLC analysis was carried out using an Agilent 1260 series. Separations were conducted by use of an Eclipse C18 column , ALE group; AFB1 at the concentration of 72 μg/kg, bm , AFB1 group; and AFB1 plus ALE, ALE+AFB1 group. Animals were orally gavaged daily with the respective doses for 42 consecutive days. The initial and final body masses of male rats were recorded in each treatment group, and they did not show any significant change among the treated groups.Rats were allowed to acclimatize to their environment for two weeks before the experiment started. They were randomly divided into five treatment groups of five rats each and treated with sterile water, control group; 4% DMSO (1 mL/kg body mass), which is the solvent for AFBg for 20 min. Blood plasma was kept at −80°C until analysis. Brains were removed, released from adhering fat and connective tissues, washed with chilled saline solution (0.9%), dried on tissue paper, and mass determined. A portion of the brain was immediately kept in 10% formalin for histological studies. Another portion of 2 g of the brain was minced and homogenized , separately, in ice-cold phosphate buffer  in a Potter–Elvehjem type homogenizer. Homogenates were centrifuged at 10,000 × g for 20 min at 4°C to pellet cell debris, and the supernatant was collected and stored at −80°C for further analyses.Animals were euthanized at the end of the experimental period, by use of isoflurane  by inhalation. Blood was collected by cardiac puncture in test tubes containing heparin. Plasma was obtained from whole blood by centrifugation at 860 × The tests were performed using a UV-visible spectrophotometer.The plasma lipid profile was performed by measuring concentrations of total lipid (Cat. No. TL20 10), cholesterol (Cat. No. CH12 20), triglycerides (Cat. No. TR20 30), high-density lipoprotein (HDL) (Cat. No. CH12 30), and low-density lipoprotein (LDL) (Cat. No. CH12 31). Analyses were done by colorimetric methods using commercial kits . Concentrations of VLDL in blood plasma were calculated according to Friedewald's equation [β-cells (HOMA-B) provided valid and reliable information based on the fasting plasma glucose level and insulin concentration. HOMA-IR and HOMA-B were calculated with a formula adapted by Matthews et al. [Concentrations of glucose were quantified by use of a colorimetric method using a commercially available kit . Concentrations of insulin were measured by ELISA with the assay kit according to the manufacturer's instructions . The homeostasis model assessment of insulin resistance (HOMA-IR) index and the function of the pancreatic s et al. .α), indoleamine-pyrrole 2,3-dioxygenase (IDO), and tissue inhibitor metallopeptidase (TIMP3) were obtained from Bioneovan Ltd., Beijing, China.ELISAs were performed for these parameters as per the manufacturer's instructions. The ELISA kits for tumor necrosis factor-alpha (TNF-Concentrations of thiobarbituric acid reactive substances (TBARS) (Ref. MD25 29), uric acid (UA) (Ref. UR21 20), and nitric oxide (NO) (Ref. NO25 33) and the activity of superoxide dismutase (SOD) (Ref. SD25 21), glutathione S-transferase (GST) (Ref. GT25 19), and glutathione peroxidase (GPx) (Ref. GP25 24) in brain supernatants were determined spectrophotometrically by use of commercially available assay kits obtained from the BioDiagnostic Company . Measurements of the above parameters were performed strictly according to the manufacturer's instructions. Urea was measured in the plasma by the urease-Berthelot method   = ∆A × total volume × 1000/3.2 × brain supernatant volume. The absorbance was measured at 250 nm against air after 30 s and 90 s.AChE activity was estimated according to Ellman's method . It was n et al. , where 11 influence on tissue structure compared to the control and ALE treatment alone. Combined treatment using AFB1 and ALE (ALE+AFB1 group) has also been assessed to evaluate the potential beneficial effects of ALE on the brain. Brain tissues, fixed in 10% formalin immediately after collecting, were processed by cutting pieces in tissue cassettes with an automated tissue processor. They were then embedded in paraffin and, after that, were sectioned with microtome at 4-6 mm thickness. Slides were then stained with hematoxylin and counterstained with eosin for histological examination. Photographs were taken on an Olympus XC30 microscope (Germany) with a digital camera (Olympus UC30 camera). Representative pictures were taken at 20x and 40x magnification from each group.The brain sections were assessed histologically, using the H&E staining method, to provide general knowledge concerning the potential aflatoxin Bp < 0.05), and the differences between experimental groups were investigated. All parameters were expressed as the mean ± SD in the figures. Principal component analysis (PCA) was also performed for all measured parameters.Statistical analysis was performed using Statistica 13.1. All biochemical measurements were performed in 5 replicates. The distribution of the data (the Kolmogorov-Smirnov and Shapiro-Wilk tests) and homogeneity of variance (the Levene test) was checked before analysis. The data fulfilled analysis of variance criteria; therefore, parametric tests were used: ANOVA  decrease in total plasma lipids (412.0 ± 12.2 mg/dL), cholesterol (105.3 ± 1.8 mg/dL), triglycerides (139.4 ± 5.8 mg/dL), low-density lipoprotein , and very-low-density lipoprotein  compared to control . Simultaneously, a significant (p ≤ 0.05) increase in the concentration of high-density lipoproteins (HDL: 44.4 ± 2.9 mg/dL vs. control: 33.8 ± 3.2 mg/dL) in the plasma of rats from the ALE group was found . The only exception was found for HDL (25.6 ± 2.1 mg/dL), where a significant decrease in concentration in the AFB1 group was noted compared to the rest of the experimental groups (1 (ALE+AFB1 group) reduced the concentration of total lipids (537.0 ± 12.6 mg/dL), cholesterol (164.9 ± 12.8 mg/dL), HDL (34.4 ± 2.7 mg/dL), and LDL (107.5 ± 10.7 mg/dL) to the values typical for the control group  and total cholesterol. A reduction of cholesterol and triacylglycerols concentrations by 44% and 58% was observed in obese, hyperlipidemic, and insulin-resistant Zucker rats, infused with CGA at a dose of 5 mg/kg body weight/day for three weeks [α). This nuclear receptor acts as a transcription factor regulating the expression of genes related to metabolism including lipid metabolism [When studying the brain, lipids are of particular interest because they account for more than 50% of its dry mass. They are the main components of cell membranes and are repositories of chemical energy. The crucial role of lipids in brain physiology and cell signaling has been demonstrated by neurologic disorders and several neurodegenerative diseases in which lipid metabolism is altered . Previouee weeks . One of ee weeks . More retabolism . Our res1 alone resulted in different responses. Treatment with ALE alone caused no significant effect on insulin concentration (4.4 ± 0.2 μU/mL) and the homeostatic model assessment for insulin resistance  index, but the concentration of glucose (84.2 ± 1.0 mg/dL) was slightly less, and the homeostatic model assessment for beta cells  index was elevated compared to the control group  (1 resulted in significantly greater concentrations of glucose (116.4 ± 4.8 mg/dL) and insulin (4.8 ± 0.1 μU/mL) in blood plasma of rats and the HOMA-IR index (1.38 ± 0.1) compared to rats in all other groups. Furthermore, in rats exposed to AFB1, the HOMA-B index (14.9 ± 0.8) was significantly less than in the other groups. The treatment of AFB1-exposed rats with ALE restored the levels of glucose (97.6 ± 5.6 mg/dL), insulin (4.2 ± 0.2 μU/mL) and HOMA (HOMA-IR: 1.0 ± 0.1 and HOMA-B: 15.7 ± 1.1) indices.Exposure to ALE or AFBctively) . Aflatox1 increased insulin resistance, as shown by the decrease in HOMA-B values associated with elevated HOMA-IR values, which corresponds to decreased insulin effectiveness in regulating plasma glucose. However, simultaneous exposure to ALE and AFB1 positively affects the discussed parameters and brings them to the control level.Insulin is pivotal for maintaining glucose metabolism in the periphery and the brain . AlteratThe positive effect of artichoke extract can be again associated with the impact of chlorogenic acid. Mentioned above studies by Rodriguez de Sotillo and Hadley  on Zuckeα , indoleamine-pyrrole 2,3-dioxygenase , and tissue inhibitor metallopeptidase   increase in TNFα (2387.5 ± 31.8 pg/mg protein) and IDO (2334.5 ± 129.4 pg/mg protein) and, at the same time, a decrease in concentrations of TIMP3 (773.0 ± 45.2 pg/mg protein) compared to controls . These evaluated parameters were generally positively influenced by the administration of AFB1 together with ALE (ALE+AFB1 group). The concentration of TNFα value (1658.5 ± 30.4 pg/mg protein) in rats exposed to ALE+AFB1 was less than that found in the rats exposed to AF1 alone but was still greater than in the control group. The mean IDO value (1726.0 ± 74.7 pg/mg protein) in brains of rats exposed to ALE+AFB1 was similar to that of the control group (1542.7 ± 41.9 pg/mg protein). Concentrations of TIMP3 (2274.0 ± 142.8 pg/mg protein) in brains of rats exposed to AFB1 and ALE simultaneously were greater than in those exposed to AFB1 alone (779.0 ± 45.2 pg/mg protein), but still significantly less compared to unexposed rats in the control group (3821.0 ± 74.9 pg/mg protein).Treatment of rats with ALE had no significant influence on concentrations of tumor necrosis factors protein) . Aflatoxα is a proinflammatory cytokine that is upregulated in response to various injuries in the hippocampi of the ischemic brain [1 in the present study affects the concentrations of various inflammatory markers in brains of rats, including proinflammatory cytokines (TNF-α) and tissue inhibitor of metalloproteinases (TIMP3). Results of previous studies have provided evidence that TNF can protect neurons by encouraging the expression of antiapoptotic and antioxidative proteins [1 (ALE+AFB1) confirmed the beneficial effect of ALE on these parameters by alleviating the toxic damage caused by AFB1 alone and restoring their normal values.TNF-ic brain  and chroic brain . Consistproteins . Furtherproteins , similarproteins . CombineκB), IL-1β, and TNF-α. Our research shows that artichoke extract, rich in phenolic acids, has neuroprotective effects through its influence on pro-inflammatory proteins.Phenolic acids, also CGA, can reduce tissue inflammation and apoptosis of nerve cells and stimulate brain cell protection . The lat1 led to threefold increase in concentration of TBARS (34.8 ± 2.34 nmol/mg protein), ALE caused a significant reduction of this parameter (8.84 ± 0.88 pg/mg protein) in brains of rats compared to control (11.8 ± 0.8 pg/mg protein) (1 and ALE (ALE+AFB1) reduced concentrations of TBARS (16.7 ± 1.4 pg/mg protein), compared to the aflatoxin B1-treated group (34.8 ± 2.34 pg/mg protein). However, the concentration remained greater than in the control group. The xanthine oxidase (XO) activity and concentration of uric acid (UA) were consistent with the TBRAS pattern and were significantly greater in rats exposed to AFB1 (XO: 67.9 ± 2.7 μmol/h/mg protein and UA: 4.7 ± 0.4 mg/mg protein), compared to controls (XO: 39.5 ± 2.9μmol/h/mg protein and UA: 2.3 ± 0.3 mg/mg protein) and all other groups  and UA concentration (2.9 ± 0.3 mg/mg protein) compared to the rats from the AFB1 group (XO: 67.9 ± 2.7 μmol/h/mg protein and UA: 4.7 ± 0.4 mg/mg protein).While aflatoxin Bprotein) . Exposur Figures . The treμmol/mg protein) and activities of superoxide dismutase (SOD: 11.6 ± 0.8 vs. control: 9.0 ± 0.7 nmol/min/mg protein), glutathione S-transferase (GST: 0.8 ± 0.04 vs. control: 0.6 ± 0.04 nmol/min/mg protein), and glutathione peroxidase (GPx: 0.21 ± 0.006 vs. control: 0.17 ± 0.02 U/mg protein). Aflatoxin B1 had the opposite effect on these parameters. After 42 days of exposure to AFB1, there was significantly less concentration of GSH (2.8 ± 0.2 μmol/mg protein) and lesser activities of SOD (5.7 ± 0.7 nmol/min/mg protein), GST (0.4 ± 0.02 nmol/min/mg protein), and GPx (0.1 ± 0.02 U/mg protein), compared to all other groups. In rats exposed to AFB1 and ALE simultaneously, the concentration of GSH (3.5 ± 0.3 μmol/mg protein) and activities of SOD (8.8 ± 0.6 nmol/min/mg protein), GST (0.6 ± 0.1 nmol/min/mg protein), and GPx (0.16 ± 0.02 U/mg protein) did not differ from values of unexposed rats   . Consistnd Koval  revealed1 leads to increased oxidative stress markers, including TBARS and decreased activities of different antioxidant enzymes, including GSH, SOD, GST, and GPx [1 caused harmful effects that cause depletion of the antioxidant defense system leading to oxidative stress and a reduction of amounts of GSH and activities of SOD and GST [1 exposure caused an obvious rise in XO levels.Results of the present study were also consistent with those of previous studies that found that treatment with AFB and GPx . Also, o and GPx . Uric ac and GPx . Consist and GST , 51. Als1 resulted in lesser activities of variable antioxidant enzymes in rats. Simultaneous use of ALE together with AFB1 brings the levels of these parameters to control ones. This result is consistent with previous studies revealing that antioxidants may protect tissue from damage by adjusting the detoxification and promoting antioxidant systems [In this study, exposure to ALE alone antioxidant content TBARS and XO levels was less than those in the control, but exposure to AFB systems , 53. Oth systems  or arsen systems . In the  systems , 55. In μmol/min/mg protein) and monoamine oxidase (MAO: 11470.0 ± 983.3 vs. control: 11049.1 ± 781.3 U/mg protein) activity. At the same time, aflatoxin B1 caused a significant increase in the activity of both enzymes (AChE: 14.5 ± 2.1 μmol/min/mg protein and MAO: 16435.8 ± 683.6 U/mg protein). AChE (9.4 ± 0.8 μmol/min/mg protein) and MAO (12890.8 ± 1578.8 U/mg protein) activity in the rats from the ALE+AFB1 group was significantly lower than those in rats exposed to ABF1 alone (AChE: 14.5 ± 2.1 μmol/min/mg protein and MAO: 16435.8 ± 683.6 U/mg protein), but still greater than that of the control group (AChE : 7.2 ± 0.5 μmol/min/mg protein and MAO: 11049.1 ± 781.3 U/mg protein)  did not affect this parameter. Exposure to both factors simultaneously (ALE+AFB1) caused a significant increase in concentrations of NO (6.0 ± 0.3 nmol/mg protein), compared to the control group (4.9 ± 0.3 nmol/mg protein)  . Neitherprotein) .1 in the brain was characterized by a significant increase in AChE activity, which is in agreement with results of previous studies [1 can be attributed to increased production of ROS that has been found to enhance the peroxidation of the plasma membrane, thereby affecting the integrity and functionality of the cholinergic system [1 significantly enhanced activity of MAO in the brain. MAO was previously shown to augment oxidative stress [1 in the present study supports the normalization of both enzyme activity to values close to the control group. The ameliorative effect of ALE may be attributed mainly to its antioxidant action and its ability to scavenge free radicals.Cholinergic dysfunction is a prime sign of neurotoxicity. Herein, the toxicity of AFB studies . Such inc system . Exposure stress . Simultaα, and IDO1, negative PC values 1 were obtained. AChE, insulin, VLDL, and triglycerides had less effect on PC 1, while NO and urea were only weakly related to it. PC2 explained a little more than 10% of the overall variability in the data. Overall, the developed PCA model (PC1+PC2) explained approximately 80% of all parameters' variability . The hippocampus consists of the twisted dentate gyrus with an open concave part directed to the hippocampus. The hippocampus is divided into three subfields: cornu ammonis 1 (CA1), cornu ammonis 2 (CA2), and cornu ammonis 3 (CA3) (1 exhibited cellular disorganization and degeneration manifested by darkly stained nuclei and extensive vacuolations with pyknotic hyperchromatic eccentric nuclei (1 group). Significantly, the treatment of AFB1-exposed rats with artichoke extract relieved these pathological changes (1 group).The brain sections were assessed histologically using the hematoxylin and eosin (H&E) staining method to determine the effects of AFB1 exhibited a decrease in the dentate gyrus's granule cell layer thickness compared to the control group. Such a decrease in thickness of the dentate gyrus's main layer results mainly from the degenerative changes of nerve cells in this area. Moreover, glial proliferation, where microglia cells' infiltration was observed, was well manifested in the sections after exposure to AFB1 exposure. The ALE+AFB1 group revealed an improvement compared to the group treated with Aflatoxin B1 alone.Histopathological assessment of the dentate gyrus in rats exposed to AFB1-induced neurotoxicity as manifested by free radicals' augmentation, cholinergic enzymes, lipid profile, TNF-α, and IDO, finds its confirmation in histopathological architecture. Degenerative changes in the hippocampus's pyramid cells and nerve cells in the dentate gyrus were observed. Moreover, inflammatory infiltrations of microglia cells and macrophages were reported. Simultaneous use of ALE with AFB1 (ALE+AFB1 group) attenuates almost all histopathological alterations induced by a single exposure to AFB1. Results of previous studies are consistent with the results observed in this study [Herein, the AFBis study –61.To sum up, differential concentration of a variety of phenolic compounds has been reported in the artichoke leaf extract capsule in this study. CGA, which is the highly concentrated component in the extract, has been found previously to have several pharmacological properties including antioxidant, anti-inflammatory, lipid lowering, hypoglycemic, antibacterial, antiviral, cardioprotective, anticancer, and immunomodulatory. Therefore, the attenuation of the adverse effects of AFB1 as well as health promotion could be attributable to the phenolic composition of the artichoke leaf extract.1 on brain function. Overproduction of free radicals manifests AFB1-induced neurotoxicity, dysregulation of lipid profile, glucose level, and insulin concentration, HOMA-IR, TNF-α, and IDO, increased activity of AChE and MAO, and inhibition of the antioxidant enzymes, HOMA-B, and TIMP3. These processes intensify oxidative stress. The factors used in this study did not have a significant effect on the urea concentration in brains of rats. Moreover, changes in histology of brain are visible after exposure to AFB1. ALE, a potent antioxidant, reduced free radical production and increased antioxidant enzyme activity. These ALE-mediated effects are significant in overcoming oxidative stress. Moreover, joint treatment using ALE and AFB1 alleviates the histopathological features induced by the exposure to AFB1. ALE can be successfully considered as a possible nominee for controlling neurotoxicity resulting from food and feed contaminated with AFB1, which causes numerous harmful effects on humans and animals' health. The phenolic compounds present in natural, balanced proportions in ALE have beneficial effects on the brain. The neuroprotective effect of ALE involves the regulation of lipid metabolism and the alleviation of inflammation and oxidative stress.Results of the study presented here demonstrate adverse effects of AFB


The device also shows an extremely low turn-on voltage (Von) of 2.2 V and a high RC of 720 mW/sr/m2/V. The results on the Von and RC of the device is demonstrated an outstanding performance among the Ir(III)-based NIR OLEDs with a similar emission peak.Near-infrared organic light-emitting diodes (NIR OLEDs) with heavy metals are regularly reported due to the advantages of their various applications in healthcare services, veil authentication, and night vision displays. For commercial applications, it is necessary to look at radiance capacity (RC) instead of radiance because of power consumption. However, recent papers still reported only simple high radiance performance and do not look at device from the point of view of RC. To overcome this hurdle, we designed Ir(III)-based heteroleptic NIR materials with two types of auxiliary ligand. The proposed emitters achieve a highly oriented horizontal dipole ratio (Ir(mCPDTiq) In particular, OLEDs are pioneering a new market beyond the visible spectral range. Recently, NIR OLEDs are of great interest not only in the field of wearable healthcare systems such as photobiomodulation (PBM)6 and photodynamic therapies (PDT)7 but also for night-vision displays and optical signal processing. In particular, phototherapy studies using OLED have been steadily published in the past decade. It is known that the effect of phototherapy is maximized in the red or NIR wavelength, where the absorption of cytochrome c oxidase (CCO), a photoreceptor, occurs well8. Recently, it has been reported that there is a wound healing effect not only in the red region, but also by using NIR OLED2. Moreover, a low level light therapy (LLLT) technique, which heals using lower energy than that of PDT, is used in PBM such as wound healing. As a result, the device was driven in a low voltage area (6 V or less) for phototherapy, and a lower voltage is required through integration, such as a battery, to develop actual commercial products. Therefore, it is more important than anything else to realize NIR OLEDs from an RC point of view. Although new NIR emitters are continuously being designed for implementation of NIR OLEDs, the performance of NIR OLEDs is still far behind devices in the visible range due to energy gap law10. Many studies have been done to overcome the bottleneck of the intrinsic performance of NIR OLEDs, such as using thermally activated delayed fluorescence (TADF)15 or using transition heavy metals such as Pt(II)20, Os(II)23, and Ir(III)30 to obtain high efficiency. The method using TADF can transfer the non-radiative triplet exciton to the singlet space and obtain 100% of internal quantum efficiency (IQE). Moreover, Pt(II) and Os(II) complexes are extremely expensive to use in comparison to the Ir(III) complex. Conventionally, many transition metal-based NIR OLEDs have been simply reported in terms of high radiance16. However, the voltage at maximum radiance is so high that there are many restrictions on applying it to actual industries from the perspective of power consumption. Recently, it has been reported that a cyclopentadithiophene (CPDT) unit has many advantages, such as efficient charge transfer, high electron density and strong electron donor ability due to a structure with high planarity in which bithiophene is fused33. Therefore, it has been adopted as a unit in photoactive layers and hole transport layers of electronic materials such as organic solar cells and quantum-dot solar cells due to its optimal properties35.OLEDs have been successfully established and are widely used as an important light source in conventional display and lighting marketsg, ≤ 2.1 eV) that is suitable for NIR emission. Furthermore, the substitution of the two methyl groups in sp3 carbon of CPDT can suppress the excessive aggregation between molecules37. In addition, we introduced two β-diketone ancillary ligands with long conjugation lengths to increase solubility and facilitate accurate wavelength control, as well as attempt to enhance the horizontal orientation39. Based on the above design concepts, the Ir(mCPDTiq)2tmd (complex 1), Ir(mCPDTiq)2acac (complex 2) were newly developed and chemical structures are shown in Fig. In this study, CPDT-isoquinoline based Ir(III) complexes for NIR OLED application were synthesized and designed because the CPDT-based ligand has an electron-rich and significant extension of the conjugative structure that enables the emission peak to have a longer wavelength. Therefore, the Ir(III) complex with a CPDT-isoqinoline ligand has a small energy band gap Eg, ≤ 2. eV that 3 nH2O) and a freshly prepared ligandisoquinoline (5)). Then, complex 1 and complex 2 were synthesized from an iridium dimer [(mCPDTiq)2Ir(μ-Cl)]2 and 2,2,6,6-tetramethyl-3,5-heptanedione (tmd), acetylacetone (acac), respectively. The synthesized iridium complexes were characterized via 1H-NMR, 13C-NMR spectroscopy and high-resolution mass spectroscopy. All intermediates were also confirmed by 1H-NMR, 13C-NMR spectroscopy and HR mass methods  showed stoutness up to 340 °C and experienced no phase change up to 250 °C. Overall, the synthesized iridium complexes exhibited sufficient thermal stability.The newly developed iridium complexes were prepared using universal Stille cross-coupling reactions between trimethylstannane (4) and 1-chloroisoquinoline. They were formed into two new iridium complexes using different ancillary ligands, as shown in Fig. 1 and 2. The electrochemical property of these Ir(III) complexes was estimated by CV experiments using tetrabutylammonium perchlorate as an electrolyte in dimethylformaide solution. The HOMO energy level was determined by comparing the oxidation potential of ferrocene, a standard material, and the synthesized materials. Both compounds 1 and 2 showed clear oxidative fluctuation at 0.78, and 0.76 V, respectively. According to the equation in Fig. 40. The band gap energy (Eg) of both compounds was same value at 2.08 eV, which was obtained from the onset value of the absorption spectra in the solution of each compounds. The LUMO energy levels were calculated by subtracting the band gap energy from the HOMO energy.A cyclic voltammetry (CV) experiment was performed to study the energy level of the newly developed complexes, as shown in Fig. 1 and 2 in dichloromethane solution and vacuum-deposited thin-film with the host material at room-temperature (298 K). Spin allowed π–π* transitions were shown to be responsible for the high absorption band below 450 nm. Relatively weak absorption bands in the range 500–700 nm in both spectra were assigned to mixed 1MLCT and 3MLCT  with an inter-ligand charge transfer (1LLCT). The photoluminescence (PL) emission peaks of complexes 1 and 2 in dichloromethane solution were observed at 760 nm with FWHM  of 79 nm and at 755 nm with FHWM of 86 nm, respectively. Complex 1 was slightly 5 nm red shifted compared with complex 2, which was due to the ancillary ligand effect. The result can be compared with the well-known already reported two iridium complexes Ir(piq)2acac (λmax = 622 nm) and Ir(piq)2tmd (λmax = 628 nm) based on the phenyl-isoquinoline ligand, and this significant red-shift was due to the electron-rich and significant extension of the conjugative structure of the CPDT unit42. These Ir(III) complexes show the absolute PL quantum yield (PLQY) and transient decay of complexes 1  and 2 , respectively  of complexes 1 and 2 were 0.80 and 0.81, which were highly horizontally oriented compared to the other organic emitters and well matched to the fitted curve, respectively. Complexes 1 and 2 were preferred to be oriented horizontally because they exhibited a heteroleptic structure. The structure of the auxiliary ligand affects the dipole moment of the emitters to be arranged horizontally, thereby enhancing the device characteristics44.To determine how the organic molecules are oriented when deposited as a thin film, angle dependent 3, 10 nm)/N,N′-Di(1-naphthyl)-N,N′-diphenyl--4,4′-diamine /Bebq2: NIR dopant /bathophenanthroline /Liq (1 nm)/Al (100 nm). ITO was patterned on a glass substrate. In the device structure, MoO3, NPB, Bebq2, Bphen, Liq, and Al were used for the hole injection layer (HIL), hole transport layer (HTL), host material, electron transport layer (ETL), electron injection layer (EIL), and cathode, respectively. In particular, Bebq2 is well known to act as an effective host during the doping process with the iridium complex and to facilitate energy transfer46.The short radiative lifetimes, efficient NIR emissions and highly horizontal oriented Ir(III) complexes prompted us to look into their EL characteristics further. The device structure of the OLEDs was as follows: indium tin oxide (ITO)/molybdenum trioxide . Moreover, all the devices were encapsulated with Al2O3 (30 nm) using the atomic layer deposition (ALD) method to measure the lifetime. The energy level and molecular structure of the materials and schematic device structure are shown in Fig. The device optimization procedure was carried out with different doping concentrations  of the NIR dopant (complexes 2) PL peak  were proved for complexes 1 and 2 at 10 wt%, respectively , light distribution characteristics and EQE are shown in Fig. ely Fig. .Table 2CBoth complexes showed the highest performance at 10 wt%, indicating that they run at significantly lower voltages compared to the previously reported NIR OLEDs, which means that a balanced charge was supplied and transported to the device as an optimal structure, resulting in direct trapping of the carrier.1 at a doping concentration of 10 wt%. The numerical values  of previous studies are presented in Table The NIR OLED developed through in study showed the best performance for complex on and the highest RC compared to the other Ir(III)-based NIR OLEDs shown in Fig. Basically, a high radiance is not very important in phototherapy using LLLT. Rather, the device must be manufactured in terms of the RC taking into consideration the driving voltage for the corresponding radiance for commercial applications. In other words, it is tremendously important to consider the power consumption of NIR OLEDs for them to be used in actual applications. The NIR OLED had an extremely low V1: 80%, complex 2: 81%) and a short radiative lifetime . Because of its high electron density and long π conjugation length, this structure can close the energy gap and emit the NIR wavelength (760 nm). Notably, the NIR OLED with our newly designed Ir(III) complex has a significantly lower Von, overwhelmingly high RC, and better reliability than previous Ir(III) complex-based NIR OLEDs. Hence, our study serves to present the future direction of NIR OLEDs for practical applications in the industry.In summary, heteroleptic Ir(III) complexes are designed and synthesized with a high horizontal dipole ratio of dipole orientation trimethylstannane (4) was prepared according to the literature49.All starting reactants were purchased from Sigma Aldrich and Alfa Aesar. The palladium catalyst (Tetrakis(triphenylphosphine)palladium(0)) was purchased from Umicore. Iridium hydrate was supplied by Furuya Metal Korea. All commercially purchased reagents were used without any further purification, and a series of intermediates were synthesized according to the reported method1H NMR and 13C NMR) were measured using a 300 MHz (Bruker) and a 500 MHz (DRX) spectrometer. Mass spectra (FAB and EI data) were acquired with a high-resolution mass spectrometer using a JMS-700 (Jeol). To analyze the thermal stability of prepared iridium complexes, thermogravimetric analysis was conducted using TA 2050 TGA instruments. All differential scanning calorimetry (DSC) data were measured using a TA Instruments 2100 DSC and heated from room temperature to 800 °C (10 °C/min.) Ultraviolet–visible absorption spectra were acquired on a LAMBDA-900 spectrophotometer (PerkinElmer). Cyclic voltammetry (CV) was measured to study the electrochemical properties of the prepared materials. A three-electrode system was used with a carbon glass electrode, an Ag/AgCl electrode and platinum wire as the working electrode, reference electrode and counter electrode, respectively. For detailed experimental conditions, a cyclic voltammogram was recorded using tetrabutylammonium perchlorate as an electrolyte in a 0.1 M dimethylformamide solution under nitrogen gas at a scan rate of 50 mV/s.All spectra : δ (ppm) = 7.07 , 7.02 .This reaction was done following the literature method48. 1H NMR : δ (ppm) = 7.21 , 7.12 , 3.5 .This reaction was done following the literature method48. 1H NMR : δ (ppm) = 7.19 , 7.02 , 1.48 .This reaction was done following the literature method49. 1H NMR : δ (ppm) = 6.98 , 6.90 , 6.84 , 1.27 , 0.31—0.12 .This reaction was done following the literature method3)4, 1.1 g, 1.0 mmol) was added to the reaction mixture and stirred at 105 °C for 12 h. After cooling to room temperature, the reactant mixture was quenched by water and it was extracted with methylene chloride three times. Then the organic solution portion was dried using anhydrous magnesium sulfate and evaporated. After solvent was removed, the product was purified by column chromatography using hexane and ethyl acetate . 1H NMR : δ (ppm) = 8.54 , 8.40 , 7.80 , 7.65–7.54 , 7.47 , 7.19 , 6.99 , 1.46 . 13C NMR : δ (ppm) = 161.42, 161.10, 153.41, 143.63, 142.07, 138.06, 137.24, 135.31, 129.99, 127.64, 127.29, 126.42, 126.35, 125.54, 122.84, 120.95, 119.23, 52.71, 45.38, 24.94 HRMS-EI + (m/z): [M] + calcd for C20H15NS2, 333.06; found, 333.0648.trimethylstannane , 1-choloroisoquinoline  were dissolved in toluene (500 mL) and purged with nitrogen gas for 30 min. Then a palladium catalyst isoquinoline (5)  and iridium(III) chloride hydrate  were dissolved in ethyl glycol and distilled water. The reaction mixture was stirred overnight under nitrogen gas. Then, the precipitated solids were filtered and washed with water and a small amount of diethyl ether. These crude products were thoroughly dried and then used without a further purifying procedure.2Ir(μ-Cl)]2 , 2,2,6,6-tetramethyl-3,5-heptanedione  and sodium carbonate  were dissolved in ethyl glycol. The three-neck flask was refluxed for 12 h at 120 °C, and then the cooled reaction mixture was poured into iced water and the precipitated solid was filtered and the filtered solids were extracted with dichloromethane and water. The organic portion was dried using anhydrous magnesium sulfate and evaporate under reduced pressure. After removal of the organic solvent, the black-purple product was purified by column chromatography using hexane and ethyl acetate . 1H NMR : δ (ppm) = 9.01–8.98 , 7.97 , 7.87–7.83 , 7.75–7.71 , 7.16–7.14 , 7.12 , 5.51 , 1.58 , 0.79 . 13C NMR : δ (ppm) = 194.34, 167.44, 165.19, 164.68, 149.95, 141.86, 141.67, 138.16, 136.92, 133.62, 130.81, 127.19, 126.98, 126.57, 126.43, 124.15, 120.68, 114.50, 93.57, 91.89, 91.57, 89.98, 86.87, 85.78, 85.41, 54.14, 53.9, 53.78, 53.63, 53.42, 53.26, 53.21,53.06, 52.70, 45.78, 40.90, 27.57, 23.99, 22.91. HRMS-FAB + (m/z): [M + H] + calcd for C51H47IrN2O2S4, 1040.21; found, 1040.2174.A prepared μ-chloro-bridged dimer  + calcd for C45H35IrN2O2S4, 956.1211; found, 956.1216.(Yield: 34%) −6 torr). The thickness of the complexes was measured using a quartz thickness-monitor. Then, UV–vis absorption and PL spectra were measured using a Fluorolog-3 (HORIBA). The excitation wavelength was set to 430 nm in both complexes. Moreover, the PLQY of the Ir(III) complexes was measured using a Fluorolog-3 (HORIBA).Thin-films (50 nm) were vacuum deposited on a glass substrate under a high vacuum (2 2)-filled glove box to avoid degradation from the air. A full angle dependent p-polarized PL spectrum was obtained with the goniometer based motorized intensity measurement system . Simulation and fitting with the experimental data were made with a customized MATLAB code describing the coherent dipole radiation theory.The emission layer was deposited on a bare 50 nm-thick glass substrate. Then, glass encapsulation was done in a nitrogen  and guest (complexes 1 and 2) were co-deposited at various doping concentrations under a high vacuum of 2 −6 torr. All the devices were encapsulated within Al2O3 using the atomic layer deposition (ALD) process under 70℃. The current density–voltage-radiance (J–V–R) and angle-resolved EL intensity characteristics of the fabricated devices were obtained with a source-measure unit (Keithley 2400) using a calibrated photodiode  and a fiber optic spectrometer  held on a motorized goniometer  in a nitrogen (N2)-filled glove box. The EQE (ηEQE) and PE (ηPE) of the devices were estimated from the measured full angular characteristics without Lambertian simplification50. The operating lifetime was monitored using a McScience Polaronix M6000 OLED Lifetime Tester.All organic and inorganic materials were purchased from Sigma-Aldrich and iTASCO. The devices were fabricated on a patterned ITO glass substrate with a sheet resistance of 15 Supplementary Information.