More than 20 unique diseases such as diabetes, Alzheimer’s disease, Parkinson’s disease are caused by the abnormal aggregations of pathogenic proteins such as amylin, β-amyloid (Aβ), and α-synuclein. All pathogenic proteins differ from each other in biological function, primary sequences, and morphologies; however, the proteins are toxic when aggregated. Here, we investigated the cellular toxicity of pathogenic or non-pathogenic protein aggregates. In this study, six proteins were selected and they were incubated at acid pH and high temperature. The aggregation kinetic and cellular toxicity of protein species with time were characterized. Three non-pathogenic proteins, bovine serum albumin (BSA), catalase, and pepsin at pH 2 and 65 °C were stable in protein structure and non-toxic at a lower concentration of 1 mg/mL. They formed aggregates at a higher concentration of 20 mg/mL with time and they induced the toxicity in short incubation time points, 10 min and 20 min only and they became non-toxic after 30 min. Other three pathogenic proteins, lysozyme, superoxide dismutase (SOD), and insulin, also produced the aggregates with time and they caused cytotoxicity at both 1 mg/mL and 20 mg/mL after 10 min. TEM images and DSC analysis demonstrated that fibrils or aggregates at 1 mg/mL induced cellular toxicity due to low thermal stability. In DSC data, fibrils or aggregates of pathogenic proteins had low thermal transition compared to fresh samples. The results provide useful information to understand the aggregation and cellular toxicity of pathogenic and non-pathogenic proteins. Misfolded or unfolded proteins under unfavorable environmental conditions are prone to form aggregates via hydrophobic interactions of proteins4. Soluble proteins are converted into insoluble aggregates that cause cellular toxicity5. In amyloidosis, insoluble protein aggregates are deposited extracellularly and they leads to tissue damage and diseases6. Pathogenic proteins in amyloidosis include lysozyme, Aβ, insulin, superoxide dismutase (SOD), α-synuclein, amylin, and β2-macroglobulin9. Although all proteins differ from each other in biological function, primary sequences, and morphologies, pathogenic proteins aggregates share many common features such as insolubility, protease resistance, and optical behavior with certain dyes such as thioflavin T (ThT)11.More than 20 different human diseases such as Alzheimer’s disease, Parkinson’s disease, and type II diabetes are associated with protein misfolding and aggregation12. Other databases such as the Human Protein Atlas have antibody findings for more than 12,000 protein-coding genes13. Although there are lots of proteins in the human body, not all of human proteins are amyloidogenic or pathogenic. Protein folding and aggregation are affected by various factors such as isoelectric point (pI), pH, ionic strength, temperature, protein concentration and the secondary structure of proteins14. The isoelectric point (pI) is the pH of a solution at which the net charge of a protein becomes zero. Protein solubility is minimal at its pI due to minimal protein charge–charge repulsions. Amyloid-like fibril formation is often seen at pH values away from PI15. Ionic strength and pH are other key solution conditions, which alter charge–charge interactions. High temperatures disrupt hydrogen bonding and hydrophobic interactions of proteins, which leads to protein denaturation and aggregation. The effect of protein concentration on protein aggregation has been investigated extensively14. The increase in protein concentration enhances the formation of protein aggregates due to increased chance of collision16. The secondary structure transition from α-helices to β-sheets also precedes aggregation of proteins. In this research, we selected six proteins, bovine serum albumin (BSA), catalase, pepsin, insulin, SOD, and lysozyme, and we investigated similarities and differences in protein aggregation kinetics, cellular toxicity, and morphological structures of six proteins.Human body generates many different kinds of proteins. According to the Human Proteome Project (HPP), the database has 19,467 predicted protein-coding genesHere is the basic information of six proteins that are selected for this research. This is first step to understand the protein aggregation of proteins.17. BSA molecule consists of 583 amino acids and the molecular weight of BSA is 66.4 kDa18. The amino acid chain is made up of three homologous domains  and each domain consists of two sub-domains, A and B20. Secondary structure is important for enzymatic function and protein aggregation. The secondary structure of BSA is mainly α-helical (74%) and the remaining polypeptide chain is in turns21. BSA has abundant ionic amino acids such as glutamic acid and lysine, while it has low tryptophan, methionine, glycine and isoleucine content. The isoelectric point of bovine serum albumin is 4.821.Albumin is the most abundant protein in plasma. Bovine serum albumin (BSA) is a globular protein and biological function of BSA includes the delivery of nutrients to cells, balancing plasma pH, and solubilization of fatty acids. Due to its stability, BSA is used for numerous biochemical applications such as drug delivery systems24. The beef liver catalase monomer consists of 506 amino acids with one heme group and one NADH molecule. Catalase is a tetramer of four identical holo monomers25. Each monomer has 26.4% α-helices and 21.7% β-sheet in secondary structure26. Irregular structure including single stands and loops plays a major role in the assembly of the tetramer. Isoelectric point of the protein is 6.727.Catalase (EC 1.11.1.6) is an antioxidant enzyme that decomposes hydrogen peroxide to water and oxygen28. Porcine pepsin consists of 327 amino acids, which has a molecular weight of 34,620 g/mol29. Isoelectric point of the porcine pepsin is between 2.76 and 2.9030. The secondary structure of pepsin is 14% helices and 44% β-sheet. Ten α-helices encompasses 46 residues and 32 β-strands encompasses 144 residues31. There are random coils as well.Pepsin (EC 3.4.23.1) is an enzyme that breaks down proteins into smaller peptides. It is one of the main digestive enzymes produced in the stomach. The maximal optimal pH for the pepsin is 1.5–2.52·−) radical into oxygen (O2) and hydrogen peroxide (H2O2)32. There are three isoforms, known as SOD1, SOD2, and SOD333. SOD1 is the cytosolic copper (Cu)-zinc (Zn) dimeric form, SOD2 is the mitochondrial manganese SOD tetrameric form, and SOD3 is the extracellular Cu, Zn tetrameric form. Mutations of SOD1 gene have been associated with amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease34. SOD1 has a molecular weight of 32 kDa with one copper- and one zinc-binding site per 153-amino acid subunit. SOD2 and SDP3 have molecular weights of 92 kDa and 135 kDa respectively33. The secondary structure of SOD1 is 4.4% helices and 29.9% β sheets35. The isoelectric point of human SOD1 is 6.3, 6.0, 5.7, and 5.0 for four isoforms36.Superoxide dismutase  is an enzyme that converts superoxide  and B (30 residues)39. The A chain contains an N-terminal α-helix (residues A1-A8), turn in the middle, and C-terminal helix (A12-A19). The B chain contains an N-terminal strand (B1-B5), β-turn (B6-B9), central α − helix (B9-B19), second β-turn (B20-B23), and C-terminal β-strand (B24-B28)40.Insulin is a hormone that allows the blood glucose to enter cells and a deficiency of the insulin or inefficient use of the insulin causes diabetes41. Mutations in human lysozyme can cause protein misfolding and large quantities of lysozyme aggregates can be accumulated in liver, kidney, and other regions of gastrointestinal tract43. Due to lots of information available on lysozyme aggregation coupled with the multiple conditions, lysozyme has been used as a model protein for amyloid fibril formation9. Hen egg white lysozyme (HEWL) is homologous to human lysozyme that is responsible for non-neuropathic systemic amyloidosis and the aggregation of HEWL stimulates cellular toxicity. HEWL is a single polypeptide chain and the molecular weight of HEWL with 129 amino acids is 14.3 kDa44. HEWL has four intramolecular disulfide bridges and an isoelectric point near 11.35. HEWL has four α-helices, a 310-helix, and a triple-stranded anti-parallel β-sheet with four disulphide bonds45. The lysozyme aggregation has been studied extensively in vitro. Since there is no net charge of lysozyme near isoelectric point, pH 11.35, lysozyme precipitation was observed over pH 11. For example, HEWL (4–200 μM) at pH 12.2 aggregates without a lag phase at room temperature (25 °C)46. HEWL also forms fibrils in acidic pHs. HEWL was incubated at either 37 °C or 65 °C with either pH 2.0 or 4.042. Fibril formation was observed in all conditions; however, the fastest rate of fibril formation was at 65 °C with pH 2. HEWL above the critical concentration, 50 μM, also aggregates at pH 1.6 and 65 °C. Protein denaturants such as ethanol and guanidine hydrochloride were also used for lysozyme aggregation study. HEWL can serve as a model protein to investigate lysozyme amyloidosis.Lysozymes (EC 3.2.1.17) are a family of enzymes with antimicrobial activity by breaking down bacterial cell walls47. Unfolded proteins induced by unfavorable environments are prone to form aggregates via hydrophobic interaction between proteins. At pH 2, hen egg white lysozyme (HEWL) aggregates faster than other acidic pHs such as pH 3 and pH 442. The aggregation process of HEWL can be accelerated when the protein is at higher temperatures 42. The aggregation kinetics also depends on the protein concentration. Higher concentration (3.0 wt %) of HEWL reduces the lag time in protein aggregation than lower concentrations (1.0 wt % or 2.0 wt %), and higher concentration also enhances the light scattering intensity42. In this research, we dissolve six proteins, BSA, catalase, pepsin, insulin, SOD, and lysozyme in pH 2 solution with 150 mM NaCl and we incubate them at 65 °C. In order to understand the effect of protein concentration on the kinetics of protein aggregation, two concentrations (1 mg/mL and 20 mg/mL) of proteins are selected. We will investigate protein aggregation kinetics, cellular toxicity, and morphological structure of six proteins in this condition.Proteins are susceptible to environmental changes such as temperature, pH, and denaturantsAll six proteins, bovine serum albumin (BSA), catalase, pepsin, lysozyme, insulin, and superoxide dismutase (SOD), were purchased from Sigma-Aldrich . All other chemicals, unless otherwise specified, were obtained from Sigma-Aldrich.Lyophilized proteins (1.0 mg or 20.0 mg) were freshly dissolved in 1.0 mL of pH 2 solution with 150 mM NaCl. The solution in 1.5 mL eppendorf tubes was incubated at 65 °C under a quiescent condition and samples were taken out at designated time points. For cell viability assays, RPMI 1640 medium adjusted to pH 2 was used instead of pH 2 solution.Thioflavin T, a benzothiazole salt, is widely used to quantify the protein aggregates. Protein samples (80 μL) was mixed with 80 µL of ThT solution (30 µM) in a 96-well plate. After 20 minute incubation, the ThT fluorescence intensity of each sample was measured by excitation at 440 nm and emission at 490 nm, using an Infinite® 200 Pro microplate reader .ANS  was dissolved at 0.1% w/v in Na-K phosphate buffer . Protein samples (140 µL) were incubated with 15 µL of ANS stock solution and the fluorescence of ANS was excited at 385 nm and the emission intensities were observed at 500 nm. A 20 μL sample was also withdrawn for protein concentration determination using the Bradford protein assay.® CRL-1721™) cell lines from the American Type Culture Collection (ATCC)  were maintained at 37 °C under a humidified atmosphere of 5% CO2. PC-12 cells were maintained in RPMI-1640 medium containing 5% (v/v) fetal bovine serum and 10% (v/v) heat-inactivated horse serum, supplemented with 1% penicillin (100 U/mL) and streptomycin (100 µg/mL).PC-12 (ATCC4 cells/well in 96 well plates) in RPMI 1640. The cells were treated with protein samples for 24 hours followed by the MTT assay. Next, 10 μL of MTT solution (5 mg/mL in PBS) was added to each well, and the cells were incubated for 4 hours. Yellow MTT solution is reduced to purple formazan in living cells and the absorbance of formazan crystals was measured at 570 nm (λabs = 570 nm) using an Infinite® 200 Pro microplate reader. Percentage cell viability was calculated by comparing the absorbance of the control cells (cells with medium only without any protein aggregates) to that of protein aggregates-treated cells (sample). Percentage cell viability = (sample with MTT – sample)/(control with MTT – control) × 100.An MTT -2,5-diphenyltetrazolium bromide) reduction assay was performed to measure the toxicity of protein aggregates. Protein samples were prepared as described in the protein sample aggregation section. At designated time points , protein samples were neutralized to pH 7.4 and they were added to PC-12 cells  were incubated at 65 °C for 2 hours. Then, 20 μL of protein solution were placed on carbon-coated copper grids , and the protein samples on the grid were negatively stained with 2% aqueous ammonium molybdate. After sufficient air-dry, grids were examined in a FEI Tecnai transmission electron microscope (TEM) at an accelerating voltage of 200 kV.48.Six proteins, BSA, catalase, pepsin, lysozyme, insulin, and SOD, in pH 2 solution (1.0 mg/mL) were incubated at 65 °C for 2 hours. Secondary structure of fresh proteins at pH 7.0 or proteins at pH 2.0 for 2 hours was measured using a Jasco model J-815 circular dichroism (CD) spectropolarimeter . CD spectra in the far UV range (190–260 nm) were obtained using a 2 mm quartz cell and a Xe lamp as a light source. A deconvolution method (BeStSel) of CD spectrum was used to estimate the secondary structures of fresh proteins and protein aggregates at pH 2.0 for 2 hoursEach bar graph represents a mean ± standard deviation of at least three independent experiments. Statistical analysis was performed using Student’s t-test, comparing each treatment to cells only unless otherwise mentioned. In all statistical analysis p < 0.05 (*) was considered significant.2. Protein aggregation is influenced by physicochemical and biological factors such as temperature, pH, and salt concentration49. However, what leads to protein toxicity has not been clearly elucidated.Protein may cause cellular toxicity when aggregated. Fibrillar aggregates of proteins are associated with more than 20 degenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and type II diabetes respectivelySix proteins, lysozyme, insulin, superoxide dismutase (SOD), bovine serum albumin (BSA), catalase, and pepsin, were selected and similarities and differences in aggregation mechanisms were investigated in this research. Three proteins, lysozyme, insulin, superoxide dismutase (SOD), were in the list of pathogenic proteins. Catalase, an antioxidant enzyme, was selected as a counterpart of SOD, another antioxidant enzyme. Pepsin, a digestive enzyme, was chosen as a counterpart of lysozyme, an antimicrobial enzyme. BSA, the most abundant protein in plasma, was used for a counterpart of insulin, a hormone that regulates the blood glucose level.In Fig. 51. ANS enhances fluorescence signals as it binds to hydrophobic regions on the protein surface53. Therefore, the ANS method has been used to detect transient states in protein denaturation. The fluorescent properties of ThT have also been well studied. ThT is used to investigate the formation of amyloid fibrils, as ThT does not bind to proteins in the folded, completely unfolded and partially folded or denaturated states of the type of a melted globule, as well as with amorphous aggregates of proteins55. Both amyloid fibrils and amorphous aggregates enhance the signal of light scattering; however, only amyloid fibrils increase the ThT fluorescence55. In protein aggregation, unfolded proteins could be formed by unfavorable environments, and the unfolded proteins are prone to form amorphous aggregates or fibrils via inappropriate interaction with other proteins since hydrophobic regions are exposed to the hydrophilic cell environment. For the protein aggregation in this research, both ANS and ThT fluorescence were monitored to understand the kinetics of protein denaturation and amyloid fibril formation.In this research, two fluorescent dyes, ThT and ANS, have been applied to characterize protein denaturation and protein aggregation. ANS is weakly fluorescent in hydrophilic environment such as water; however, its intensity is increased in hydrophobic environment such as nonpolar solvents® CRL-1721™) that was a cell line derived from a pheochromocytoma of the rat adrenal medulla. After 24 h of incubation, cellular viability of protein aggregates was measured by the reduction of MTT -2,5-Diphenyltetrazolium Bromide). Fresh or aged BSA, catalase, and pepsin did not induce any cytotoxicity to PC-12 cells. There was no statistical difference in MTT reduction between cells only in medium and cells treated with fresh or aged BSA, catalase, and pepsin. Other three proteins, lysozyme, insulin, and SOD, were not toxic when they were fresh; however they induced the cytotoxicity when incubated for more than 30 minutes. For insulin, protein for 30 minute incubation caused 45.8 ± 12.6% toxicity to PC-12 cells and insulin for 120 minute incubation increased the cytotoxicity to 63.6 ± 4.2%.In Fig. Transmission electron micrograph (TEM) was used to visualize protein species formed at pH 2 and 65 °C for 2 hours. In Fig. 57. Cytotoxicity of Aβ may be maximal for the oligomers of intermediate size58. From monomer or dimer, cytotoxicity increased with oligomer size; however, cytotoxicity decreased with size when Aβ oligomers are bigger than 12-mers or higher60. In this research, other two pathogenic proteins, SOD and lysozyme, at pH 2 and 65 °C for 120 minutes forms protofibrils or premature fibrils  is one of useful tools to measure thermal denaturation of proteins. In Fig. Circular dichroism (CD) spectroscopy has been used to examine secondary structure changes of six proteins during aggregation. As seen in Fig. 67. The two negative bands arise from π–π* and n–π* transitions in the amide groups. Negative bands at 218 nm and positive bands at 195 nm are well-defined antiparallel β-sheets68. The conversion of the secondary structure into a predominantly antiparallel β-sheet is a pathologic process of pathogenic proteins69. In Table Protein secondary structural information can be derived from CD signals. Two negative bands at 222 nm and 208 nm and a positive band at 193 nm are characteristic α-helix CD spectra14. Proteins increase the chance of collision at higher concentrations, which enhances the formation of protein aggregates16.In order to understand the effect of protein concentration on the protein aggregation, we increased the protein concentration of six proteins to 20 mg/mL and we repeated experiments of protein aggregation kinetics, cytotoxicity, and TEM imaging. Protein concentration is one of key factors in protein aggregationHere, six proteins (20 mg/mL) were incubated at pH 2 with 150 mM NaCl and 65 °C, and the aliquots were withdrawn at the following time points . In Fig. In Fig. Transmission electron micrograph (TEM) was used again to visualize the higher concentration of protein species formed at pH 2 and 65 °C for 2 hours. In Fig. In Figs. In summary, we investigated the cellular toxicity of pathogenic or non-pathogenic protein aggregates. Three proteins, lysozyme, insulin, superoxide dismutase, were selected as pathogenic proteins, and other three proteins, catalase, SOD, pepsin, were chosen as non-pathogenic proteins. Six proteins (1 mg/mL) were incubated at pH 2 with 150 mM NaCl and 65 °C, and three pathogenic proteins, lysozyme, insulin, and SOD, enhanced ThT fluorescence emission and they reached the saturation level in 30–60 minutes. Based on TEM images and MTT test, fibrils or aggregates of three pathogenic proteins induced the cellular toxicity. Other three non-pathogenic proteins, BSA, catalase, and pepsin were stable, and they didn’t cause the cellular toxicity in this condition. According to DSC analysis, fibrils or aggregates of pathogenic proteins had low thermal transition compared to fresh samples. Low thermal stability is associated with the cellular toxicity. At a higher concentration (20 mg/mL), even non-pathogenic proteins cause cellular toxicity in the early incubation time. Pathogenic proteins induced the cellular toxicity like a lower concentration (1 mg/mL). All results in this research would be beneficial to understand similarities and differences in protein aggregation kinetics, cellular toxicity, and morphological structures of pathogenic and non-pathogenic proteins.


In the present study, we aimed to evaluate the early postoperative rotational stability of TECNIS toric intraocular lens (IOL) and analyse its correlation with preoperative and intraoperative parameters.A total of 102 eyes from 87 cataract patients who underwent implantation of TECNIS toric IOL during July 2016 to November 2017 were enrolled in this retrospective study. Preoperative parameters including corneal astigmatism, axial length (AL), lens thickness (LT), anterior chamber depth (ACD) and sulcus-to-sulcus (STS), were determined. The area of capsulorhexis was measured with Rhinoceros 5.0 software. The follow-up examinations including the residual astigmatism (RAS) and postoperative toric IOL axis, were performed at 1 month and 3 months after surgery.r = 0.522, P = 0.0003), LT  and AL . As for the area of capsulorhexis, the regressive equation was: y = 0.682 × −13.105, demonstrating that the diameter of capsulorhexis should be controlled within 5.8 mm to maintain the toric IOL rotation within 5.0°.RAS was −0.84 ± 0.88 D at 1 month and −0.81 ± 0.89 D at 3 months after surgery. The rotation of toric IOL at 3 months was 4.83 ± 3.65°. The Pearson’s r of ACD, horizontal and vertical STS, and toric IOL target axis was 0.011, 0.039, 0.045 and 0.082. The toric IOL rotation was positively correlated with the area of capsulorhexis (TECNIS toric IOLs possessed great early postoperative rotational stability. The area of capsulorhexis, AL and LT were positively correlated with postoperative rotational stability. A capsulorhexis within 5.8 mm had an important significance in improving rotational stability. As one of the main factors affecting the postoperative visual quality of patients with cataract, astigmatism may lead to visual discomfort, such as blurry vision, visual fatigue and diplopia. In accordance to the survey, patients with cataract have corneal astigmatism to varying degrees. Moreover, 21.3–22.4% of patients with cataract have 1.00 dioptre (D) to 1.50 D of corneal astigmatism, with 10.6% to 12.4% of patients having 1.50 D to 2.00 D and 8.20% to 13.0% of patients having 2.00 D or more [However, even with precise preoperative calculation and appropriate implantation of toric IOLs, residual astigmatism (RAS) still occurs mainly due to the rotation of toric IOLs. Previous studies have shown that every degree of rotation from the target axis reduces effectiveness by ~3.3%, while >30° of rotation will lead to an increase in astigmatism , 7. FurtThis retrospective study was performed at the Eye Center, the Second Affiliated Hospital of Zhejiang University. All patients underwent phacoemulsification and implantation with TECNIS toric IOLs during July 2016 to December 2017. TECNIS toric IOLs  were one-piece, hydrophobic acrylic, aspheric optic IOLs. Patients with cataract who had corneal astigmatism of over 1.0 D on corneal topography  were included in this study. The included patients must have regular corneal astigmatism on Pentacam as the astigmatism meridian of the anterior and posterior surfaces was within 10° and astigmatism power was within 0.75 D. Exclusion criteria included irregular astigmatism, such as forme fruste keratoconus, corneal scars, pterygium, traumatic cataract, glaucoma, retinal detachment, uveitis, macular degeneration or retinopathy, any experience of ocular surgery, or any other comorbidity that might affect postoperative visual function.Preoperative assessments were performed, including biometric parameters and toric IOL calculations. Preoperative biometric parameters included corneal astigmatism, axial length (AL), lens thickness (LT), anterior chamber depth (ACD) and sulcus-to-sulcus (STS).https://www.amoeasy.com/calc). A surgically induced astigmatism (SIA) was entered as 0.3 D, and the steep meridian of the cornea was used for the incision position.Keratometry was determined using the Pentacam . ACD was measured by anterior segment optical coherence tomography , while horizontal and vertical STS were determined by ultrasound biomicroscopy . Additionally, measurements of AL and LT were both performed by A-scan ultrasound. High myopia was defined as AL over 26.00 mm using A-scan ultrasound. Toric IOL power, alignment axis and meridian were calculated using an online calculation software . At the first step of surgery, toric IOL alignment axis was marked using a Mendez ring and highlighted for recognition. A clear corneal incision of 2.0 mm was made, followed by the continuous curvilinear capsulorhexis (CCC), phacoemulsification, irrigation and aspiration of the cortex. The TECNIS toric IOL was inserted and rotated clockwise to approximately 15° short of the intended axis. After viscoelastic material was removed, the IOL was precisely rotated to the target position. Finally, the accuracy of IOL placement was checked, and the incisions were hydrated. The procedures of all surgeries were recorded for further usage of the area of capsulorhexis.Patients received examinations at 1 month and 3 months after surgery. The examinations included manifest refraction, keratometry and photographic assessment of toric IOL rotation. After the pupils were totally dilated, the images of toric IOL were captured by a digital camera  connected to the slit-lamp and computer, when two symmetrically marked spots could be clearly recognized on TECNIS toric IOL.Photographic assessments included the axis of postoperative toric IOL rotation and the area of capsulorhexis during surgery. For each postoperative image, the exact axis of TECNIS toric IOL was calculated using the Adobe Photoshop software. Deviation caused by head tilt, camera rotation or other abnormity was diminished to the most extent with the help of referring to conjunctival vessels. The difference between the exact axis and the target axis demonstrated the early postoperative rotational stability of toric IOL.The area of capsulorhexis was calculated by using a 3D design software, Rhino V.5.0 . After importing the image of surgery at the step of removing viscoelastic material after implanting toric IOL, the image was resized by adjusting the optic diameter to 6.0 mm. By tracing out the capsulorhexis, the area of capsulorhexis was directly calculated. Such calculation was conducted at least three times, and the mean value was used as the final data Fig. .Fig. 1PhP values of < 0.05 were considered as statistically significant.Statistical analysis was performed by using SPSS V.11 . Variables are presented as the mean ± standard deviation. Relationships between variables were assessed using Pearson’s correlation analysis. Multiple stepwise regression analysis was applied to assess the independent effects of various factors that might be correlated with toric IOL rotation. In the present study, a total of 102 eyes from 87 patients, including 51 males and 36 females, were enrolled. The mean age of patients during surgery was 64.38 ± 13.75 years, ranging from 30 to 84 years. The mean AL was 25.17 ± 2.39 mm, while eyes with high myopia accounted for 32.35%. The mean preoperative corneal astigmatism, measured with Pentacam, was 1.80 ± 0.86 D (ranging from 1.00 D to 5.70 D). The mean preoperative toric IOL setting axis was 64.15 ± 52.03°, ranging from 0 to 178°. Table P < 0.01). RAS was 0 D in 27.45% (28/102) of patients, and it was controlled within −0.50 D and −1.00 D in 54.90% (56/102) and 87.25% (89/102) of patients at 3 months, respectively.The mean RAS was −0.84 ± 0.88 D (ranging from 0 to −2.50 D) at 1 month and −0.81 ± 0.89 D (ranging from 0 to −2.50 D) at 3 months, which was significantly reduced compared with the preoperative corneal astigmatism. No significant differences in RAS were observed between 1 month and 3 months (The mean toric IOL rotation at 3 months after surgery was 4.83 ± 3.65° (ranging from 0 to 21°). Table 2 (ranging from 21.02 to 32.65 mm2) at 3 months after surgery. Toric IOL rotation was significantly and positively correlated with the area of capsulorhexis. The regression equation was y = 0.682 × −13.105 . According to the regression equation, when the toric IOL rotation was set at 0°, the area of capsulorhexis was 19.22 mm2, corresponding to a CCC of ~4.95 mm in diameter. When the rotation was within 5°, the area of capsulorhexis was 26.54 mm2, with a CCC of 5.81 mm. When the area of capsulorhexis was over the limitation, as the diameter of CCC was over 6 mm, the mean toric IOL rotation was significantly increased to 7.81 ± 4.68° (ranging from 0 to 21°), with 66.67% (18/27) of patients over 5°. In conclusion, the diameter of capsulorhexis should be controlled within the range of 5.0–5.80 mm. Simple correlation analysis revealed that AL and LT were positively associated with toric IOL rotation (Table P < 0.01). No correlations were found between toric IOL rotation at 3 months and other variables, including ACD, STS  and toric IOL axis .The variables included in the multiple stepwise regression analysis were age, gender, AL, LT, ACD, STS , toric IOL axis and area of capsulorhexis. Only area of capsulorhexis, AL and LT were found to be independently correlated with toric IOL rotation at 3-months follow-up . We reviewed AL and LT measured before surgery and found that some patients with high myopia had LT in average as well. This result suggested that it was not advisable to only take AL in consideration. To these patients with high myopia, LT should be calculated before cataract surgery as well. Except for AL and LT, other preoperative biometric parameters, like ACD and STS, did not reveal any significant correlation with rotation of toric IOLs.In order to control the situation of axis misalignment, it is highly necessary to explore the potential factors of rotational stability. Apart from the influence of materials and shapes of toric IOLs , 20, it 2 as the maximum. The optic was completely free of contact between the anterior capsule and IOL in this case. What should be highlighted was that the other eye of this patient was also implanted with a TECNIS toric IOL 1 month prior to the surgery of this eye. In the other eye, with AL of 26.98 mm, LT of 4.47 mm and the area of capsulorhexis of 27.53 mm2, IOL misalignment was only 4° at 3 months after surgery. This case highly demonstrated that the size of capsulorhexis played an important role in risk factors of rotational stability.An appropriate size of CCC is essential to prevent toric IOL rotation. To the best of our knowledge, our present study was the first one to compare the rotational stability with the accurate size of capsulorhexis. The area of capsulorhexis was significantly associated with rotational stability. When the optic was only partially covered by the anterior capsule, the mean rotation of toric IOLs was evidently increased from 4.83 to 7.81. There was a special case in this study. The toric IOL of this patient was rotated 73° at 1 week and 81° at 1 month after surgery. The IOL repositioning was performed again around 40 days after cataract surgery, after which the IOL remained stable. In this patient, AL was a little longer as 27.07 mm, but the LT was at an average level of 4.50 mm. However, the area of CCC in this case reached 33.06 mmThere are still some limitations in this study. One is the short follow-up time since the underlying capsular shrinking probably occurs at 6 months or 1 year after cataract surgery, though it is reported within the first 3 months in the majority . AnotherOverall, a single-piece acrylic toric IOL, TECNIS toric IOL, was found to be effective in reducing pre-existing regular corneal astigmatism, and it had good early postoperative rotational stability. Moreover, the rotation of TECNIS toric IOLs was associated with LT, AL and size of capsulorhexis. A capsulorhexis within 5.8 mm had an important significance on improving rotational stability.A single-piece acrylic toric IOL, TECNIS toric IOL, showed great early postoperative rotational stability in previous studies.Although several studies reported the relationship between rotational stability and axial length, it is still unclear whether preoperative and intraoperative parameters would affect rotational stability.We found that early postoperative rotational stability of TECNIS toric IOL was correlated with axial length and lens thickness.To patients with myopia, lens thickness should be taken into account as well. An appropriate size of capsulorhexis had significantly influence on improving rotational stability of toric IOL as the diameter of 5.8 mm.


Accordingly, porous Pt showed higher oxygen reduction reaction (ORR) and oxygen evolution reaction (OER) activity than Pt black. When the activity was compared to that of a common carbon-supported electrocatalyst, Pt/ketjen black (KB), porous Pt showed a comparable ORR current density (2.5 mA cm−2 at 0.9 V for Pt/KB and 2.1 mA cm−2 at 0.9 V for porous Pt), and OER current density (6.8 mA cm−2 at 1.8 V for Pt/KB and 7.0 mA cm−1 at 1.8 V), even though the ECSA of porous Pt was only one-sixth that of Pt/KB. Moreover, it exhibited a higher durability against 1.8 V. In addition, when catalyst layers were spray-printed on the Nafion® membrane, porous Pt displayed more uniform layers in comparison to Pt black, showing an advantage in its usage as a thin layer.Porous Pt electrocatalysts have been developed as an example of carbon-free porous metal catalysts in anticipation of polymer electrolyte membrane (PEM) fuel cells and PEM water electrolyzers through the assembly of the metal precursor and surfactant. In this study, porous Pt was structurally evaluated and found to have a porous structure composed of connected Pt particles. The resulting specific electrochemical surface area (ECSA) of porous Pt was 12.4 m Such dispersion of metal nanoparticles leads to high catalytic activity, even with small amounts of noble metals. However, carbon supports are sometimes not usable. For example, in the case of PEM water electrolyzers, carbon supports are highly corroded under high potential anodic conditions over 1.8 V), which are required to obtain a practical current density of 1 A cm.8 V, whi−2 ,4. Addit−2 . Not onl−2 .−1, which is comparable to that of Pt-deposited carbon, under half-cell measurements in solution the ORR activity at 0.9 V, which is common for standard comparisons, is expected to be low [−1, has been reported [−1, and a higher durability than Pt supported on a carbon support [Even though problems related to the durability of carbon supports have been reported, increasing the electroactive surface area without a carbon support is rather challenging. A large quantity of novel metal catalysts with a low surface area should generally be employed without carbon supports, which increases their cost . For thao be low . In otheo be low ,16,17,18 support .We are rather interested in developing metal-only catalysts and improving their structure as devices. In our prior studies, mesoporous carbon supports were developed through a simple one-pot reaction that involved heating a mixture of a surfactant and carbon precursors for PEM fuel cells, and high performance and durability were achieved in accordance with their mesoporous structure ,23,24,25In this paper, porous Pt has been synthesized as one example of a porous metal catalyst by employing a Pt precursor and a surfactant. Step-by-step heat treatments were applied to reduce the Pt precursor and remove the surfactant. Resulting carbon-free porous Pt was structurally evaluated, and its electrochemical activity toward the ORR and oxygen evaluation reaction (OER) was studied in detail. Additionally, a catalyst layer of porous Pt was prepared using a spray printing method and its structure was evaluated.2 and also heat-treated at 300 °C for 1 h for decomposition of the surfactant under a nitrogen atmosphere, a sticky product was obtained owing to the residual surfactant. Therefore, the heat treatment condition for decomposing the surfactant was altered to 400 °C for 3 h. As a result, a dry powder was obtained. However, based on thermogravimetric (TG) analysis, a weight loss of 62.3% was further observed at around 200 °C 0 °C see , indicat0 °C see , resulti2 g−1, whereas that of commercially available Pt black was 14.3 m2 g−1, which was slightly low compared to the value of 25 m2 g−1 or less given by Sigma-Aldrich . Porous Pt showed mesopores consisting of less than 100 nm in diameter and micropores, while Pt black mostly exhibited small mesopores of less than 30 nm, with relatively fewer micropores. Since Pt black consists of nanoparticles not connected to each other and does not contain pores within the particles, the size of 30 nm reveals the particle size and interparticle pore size achieved by these nanoparticles.Porous Pt was characterized by nitrogen sorption in comparison to commercial Pt black. In order to view the nanostructure of porous Pt and Pt black, SEM images were observed and are shown in 7/2 and Pt4f5/2, respectively. Those two peaks indicated that both materials mostly contained the metallic Pt (0) surface [Pt 4f XPS spectra were recorded to evaluate the surface chemical states. Both porous Pt and Pt black had two peaks at 71.1–71.3 and 74.4–74.6 eV of binding energy, corresponding to Pt4f surface . Althoug surface , are appXRD patterns were also analyzed . Both po2 g−1. The ECSA of Pt/KB was 76.5 m2 g−1, which stayed at the low end of reported values (70–100 m2 g−1) [2 g−1) than porous Pt. As shown in Cyclic voltammograms of porous Pt and Pt black were recorded and are shown in  m2 g−1) ,30,31, s m2 g−1) , but por−2 for Pt/KB and porous Pt, respectively. The reported ORR activity for Pt/C is even higher, for example 3 mA cm−2 [−1 for Pt/KB and porous Pt, respectively. The reported ORR mass activity for Pt/C has the range but is mostly between 200 and 500 A g−1 [The ORR activities of all electrocatalysts were evaluated at a rotating speed of 1600 rpm and are shown in  mA cm−2 . ORR mas mA cm−2  was used00 A g−1 ,30. The −2 at 1.8 V), while Pt black revealed a lower OER current (3.4 mA cm−2 at 1.8 V) than the other two catalysts. When the reported OER activity of Pt catalysts is considered, Pt bulk and nanoparticles show an OER current density of about 4 and 10 mA cm−2 at 1.8 V, respectively [The OER activities of three electrocatalysts were evaluated at a rotating speed of 1600 rpm and are shown in ectively , which r−2, was applied to evaluate the durability in this study. Among the three samples, only Pt/KB was found to continuously lose OER current. Voltammograms of the three samples after the durability test were developed and are indicated with dotted lines in −2 under 60 °C [−2 under 80 °C [In order to evaluate durability against a high potential, the anode potential at PEM water electrolyzers was considered, since it is a more severe condition than the cathode potential at PEM fuel cells. Although the protocol for the durability examination of PEM fuel cells has been well established , the proer 80 °C  were moner 80 °C .® membrane. Pt-loading was fixed to 0.50 mgPt/cm2. The cross section of catalyst layers was observed using Focused Ion Beam (FIB)-SEM and is shown in 2 or less. The formation of thin films will be more difficult when large-scale manufacturing methods of thin layers are considered. A high void volume is also important for enhancing the electrocatalytic activity [Catalyst layers of porous Pt and Pt black were spray-printed on the Nafionactivity . Consequ2), 5 N hydrochloric acid (HCl), ethanol, 2-propanol, and 0.1 M perchloric acid (HClO4) were all purchased from Wako Pure Chemical Industries Ltd.  Pluronic® F127 was obtained from Sigma-Aldrich. 5% Nafion® dispersion solution and Nafion® 117 were obtained from Dupont . Milli-Q water was used in all cases. Those chemicals were used without any further purification.Platinum (II) acetylacetonate (Pt(acac)For the electrochemical evaluation, Pt black and Pt/KB (TEC10E50E) were used as standard electrocatalysts for the comparison in this study and obtained from Wako Pure Chemical Industries, Ltd. and Tanaka Kikinzoku Kogyo K.K. , respectively.® F127 and Pt(acac)2 were used as a template to form a porous structure and as a metal precursor, respectively. Typically, 0.4275 g of Pluronic® F127 was dissolved in a mixture of water/ethanol/HCl (2.175 g/2.875 g/75 μL). Then, 0.675 g of Pt(acac)2 was added to this solution. This mixture was stirred at 30 °C for 6 h, kept at room temperature for 6 h, and dried at 80 °C for 6 h in the oven. After further heat treatment of the resulting powder, porous Pt materials were obtained. The heat treatment conditions, such as the temperature and atmosphere, were studied to optimize the porous structure and are fully discussed in the “Results and discussion” section.PluronicFor material characterization, nitrogen sorption measurements were carried out using BELSORP-mini II-VS . Before the measurement, samples were pre-treated at 200 °C under vacuum for 2 h. The specific surface area was calculated by a BET method using the adsorption area. The Barrett Joyner Hallenda (BJH) method was also applied to estimate the pore size distribution.Thermogravimetric (TG) analyses were conducted using Thermo Plus Evo2 . Measurements were carried out in the air from room temperature to 400 °C via raising the temperature by 4 °C/min.SEM observations were performed using S-5200 , with the accelerating voltage of 30 kV. Studies with X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD) were also conducted using Kratos Axis Ultra  and RINT-UltimaIII/PSA (Rigaku), respectively.−2 in all cases. In this study, a Nafion® dispersion was not used as a binder in order to simply observe electrochemical characteristics of catalysts without a Nafion® effect. The fact that a binder is not essential for adhesion of the catalyst to the GC and that low-loaded ionomer free catalyst (≤~18 μgPt cm−2) adheres with a sufficient strength during measurements for ORR activity evaluation at room temperature has previously been reported [The dispersion containing electrocatalysts (4.3 mg), water (0.35 mL), and 2-propanol (2.56 mL) was mixed using an ultrasonic homogenizer and drop-cast onto a glassy carbon (GC) rod ), and dried at room temperature for use as a working electrode. The Pt loading was kept to 17.3 µg cmreported .4 under nitrogen atmosphere with the scan rate of 50 mV s−1. ORR and OER activities were evaluated at 1600 rpm under the saturation of oxygen and nitrogen, respectively. In this study, all potentials were converted to a reversible hydrogen electrode (RHE), and IR correction was applied for OER evaluation by actually measuring the solution resistance using a potentiostat. Furthermore, in order to observe the durability against the anodic condition of the water electrolysis, the potential of 1.8 V vs. RHE was applied for 10 min, and OER activities before and after applying such potential were evaluated.Electrochemical measurements were performed using a common half-cell set-up in the solution. A potentiostat  was used for all electrochemical measurements. Besides the working electrodes, Ag|AgCl in a saturated KCl aqueous solution and Pt wire were utilized as reference and counter electrodes, respectively. Measurements were taken in 0.1 M HClO® dispersion solution (919 µL), and MilliQ water (196 µL) was thoroughly stirred by an ultrasonic homogenizer. In this condition, the ratio of ionomer and catalyst was fixed to 0.33. This slurry was spray-printed on Nafion® 117 by a spray printing system . Catalyst layers were made into a 1 cm × 1 cm square. Pt-loading was fixed to 0.50 mgPt cm−2. The cross section of catalyst layers was observed using FIB-SEM . FIB processing was carried out at an accelerating voltage of 30 kV and a beam current of 0.4 nA.Catalyst layers were prepared by employing a common spray printing method. The slurry consisting of porous Pt (or Pt black) (81.5 mg), ethanol (1764 µL), 5% Nafion2 g−1, in comparison to a commercial Pt black electrocatalyst, which has a value of 14.3 m2 g−1. The ECSA of porous Pt was 12.4 m2 g−1, whereas that of Pt black was 4.7 m2 g−1. Accordingly, porous Pt showed higher ORR and OER activity than that of Pt black.A carbon-free porous Pt electrocatalyst was successfully prepared using the assembly of the metal precursor and surfactant under the optimized heat treatment condition. The structure of porous Pt was found to be composed of connected Pt particles with a size of 20 nm, leading to a relatively high BET surface area of 32.4 m−1 at 0.9 V for Pt/KB and 190 mA cm−1 at 0.9 V for porous Pt), ORR mass activity (230 A g−1 at 0.9 V for Pt/KB and 2.1 A g−1 at 0.9 V for porous Pt), and OER current density (6.8 mA cm−1 at 1.8 V for Pt/KB and 7.0 mA cm−1 at 1.8 V) were obtained, even though the ECSA of porous Pt was only one-sixth that of Pt/KB. However, Pt/KB lost most of its OER activity during the durability test against the anode potential during water electrolysis owing to carbon corrosion, whereas porous Pt maintained its OER activity due to the effect of the carbon-free condition.In comparison to a standard electrocatalyst, Pt/KB, an equivalent ORR current density (2.5 mA cm® membrane, porous Pt showed more uniform layers in comparison to carbon-free Pt black, demonstrating the advantage of its usage as a thin film.In addition, when catalyst layers were spray-printed on the NafionConsequently, porous Pt with high ORR and OER activities and a high durability against a high potential was successfully prepared in this study and showed potential to be used as an electrocatalyst for PEM fuel cells and PEM water electrolyzers.


To investigate the clinical features and risk factors for discerning the critical and predicting the outcome of patients with COVID‐19.Patients who were admitted to the intensive care unit (ICU) department and general infection department of TaiKang Tongji (Wuhan) Hospital from February 10 to March 27, 2020, were included. Data on clinical features, complications, laboratory parameters, chest CT, nutrient requirement, and electrolyte imbalance were analyzed retrospectively.A total of 123  patients were enrolled. 65% of patients with comorbidities, hypertension (45.5%), diabetes (21.9%), 36.5% of patients had more than one comorbidity. The proportion of lymphocytes in critical patients was significantly lower than that of non‐critical patients. The proportion of patients with increased NLR, PLR, IL‐6, CRP levels, and chest CT score was significantly higher in the critical than that of non‐critical patients. The logistic regression analysis identified low lymphocyte count, high NLR, PLR, IL‐6, CRP levels, and CT score as independent factors for discerning critical cases and high NLR, PLR, IL‐6, and CT score could predict poor clinical outcome. Furthermore, we identified patients who needed nutrition support (HR 16.99) and with correction of electrolyte imbalance (HR 18.24) via intravenous injection were more likely to have a poor outcome.The potential risk factors of lower lymphocyte count, high levels of NLR, PLR, IL‐6, CRP, chest CT score, and the statue of nutrient requirement or electrolyte imbalance could assist clinicians in discerning critical cases and predict the poor outcome in patients with COVID‐19. The potential risk factors of lower lymphocyte count, high levels of NLR, PLR, IL‐6, CRP, chest CT score, and the statue of nutrient requirement or electrolyte imbalance could assist clinicians in discerning critical cases and predict the poor outcome in patients with COVID‐19. This novel virus shares about 79% and 50% of its genetic sequence with the coronaviruses responsible for severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome (MERS).Accumulated evidence suggests that patients suffered from severe COVID‐19 with a dysregulation of the immune response.22.1We performed this retrospective study on the clinical characteristics of laboratory‐confirmed patients with COVID‐19. Patients were admitted to the intensive care unit (ICU) department and general infection department of TaiKang Tongji (Wuhan) Hospital, a major institution for COVID‐19 treatment assigned by the People's Liberation Army of China. Cases with incomplete data will be excluded. A total of 123 cases that come from the community or nursing home from February 10 to March 27, 2020 were included. All cases were positive of SARS‐CoV‐2 nucleic acid RT‐PCR and diagnosed according to guidelines of COVID‐19 in China. Critical patients and non‐critical patients (including severe and mild patients) were categorized based on the 7th edition of the Chinese National Health Commission,2.2Information about demographic characteristics, symptoms, laboratory parameters, chest computed tomographic (CT) images, and treatments was all collected and reviewed from the electronic medical record system (EMRS). Peripheral venous blood samples, which were analyzed within two h, were assessed at the central laboratory of TaiKang Tongji (Wuhan) Hospital following the standard operative procedures. The CT scans were performed in the same institution. The score of chest CT was evaluated according to the following criteria: (a) no obvious abnormality; (b) unilateral ground glass infiltration or consolidation <50% area; (c) unilateral ground glass infiltration or consolidation ≥50% area; (d) bilateral ground glass infiltration or consolidation <50% area; (e) bilateral ground glass infiltration or consolidation ≥50% area. Patients‐needed nutritional support was defined as intravenous glucose, amino acid, or albumin supplementation ≥3 days. Electrolyte imbalance was defined as requiring intravenous infusion for correcting electrolyte imbalance ≥3 days. The data used in the study were anonymous. Data of patients were checked by two independent investigators.2.3U test, χ2 test, or Fisher's exact test appropriately. The predictive value of parameters was evaluated by measuring the receiver operating characteristic curve (ROC). The optimal critical value of each parameter was determined by calculating the Youden index. Logistic regression analysis (the Enter method) was performed to determine possible risk factors associated with discerning critical patients or an outcome. P < .05 was considered statistically significant. All these statistical analyses were performed using the SPSS 23.0 or MedCalc 19.Continuous variables were appropriately expressed as median (IQR) or mean (SD). Categorical variables were presented as median (IQR) or n (%). Continuous and categorical variables were compared by the Mann‐Whitney 33.1P < .001). Patients over 60 or 75 years old between groups were also different . There was no gender difference between the two groups (P = .203). Overall, the most common initial symptoms were fever (61.7%) and cough (53.6%). 65.1% of patients had comorbidities, 36.5% of patients had more than one, including hypertension (45.5%), diabetes (21.9%), heart disease (17.1%), respiratory diseases (9.7%), chronic kidney disease (8.1%), and post‐stroke (8.1%). Of all cases, five had tumors. Patients with comorbidities in the critical group were more than in the non‐critical group (P < .000).A total of 123 patients with COVID‐19 were included in this study. The detailed information of study subjects is shown in Table 3.2The level of laboratory parameters and the score of CT, white blood cells, neutrophils, NLR, PLR, CRP, IL‐6, PCT, serum sodium concentration, PT, D‐dimer, and CT score, were significantly higher in the critical group than those of non‐critical group. On the contrary, the level of lymphocytes, uric acid, and albumin in the critical group was lower in the non‐critical group Table .P = .027, Z = 2.214, 95% CI 0.007‐0.121), and AUC of NLR was larger than AUC of PLR . There was no difference between lymphocyte and NLR (P = .082).ROC curve analysis was used to discern critical and non‐critical patients with COVID‐19. The areas under the curve (AUC) of lymphocyte, NLR, PLR, IL‐6, CRP, and CT score were 0.914, 0.965, 0.850, 0936, 0.934, and 0.930, respectively. The optimal cutoff values relative to the preceding indicators were 1.01, 4.16, 172.44, 6.49, 4.27, and 3 for lymphocyte, NLR, PLR, IL‐6, CRP, and CT score, respectively . The AUC of NLR is larger than AUC of PLR . There is no significant difference between the AUC of lymphocyte and PLR (P = .392).We further used ROC curve analysis to predict the outcome of COVID‐19 patients. The AUC of lymphocyte, NLR, PLR, IL‐6, CRP, and CT score was 0.825, 0.913, 0.788, 0.895, 0.866, and 0.823, respectively. The optimal cutoff values relative to the preceding indicators were 1.01, 4.31, 189.11, 10.63, 3.3, and 3 for lymphocyte, NLR, PLR, IL‐6, CRP, and CT score, respectively , and the adjusted OR with age and comorbidities were calculated. Results showed that seven parameters, the NLR, PLR, IL‐6, CRP, CT score, patients who need nutrition support, and electrolyte imbalance, were positively correlated with the risk of critical patients. Still, the lymphocyte count was a negative correlation Table . On the 3.4P < .000) , PLR , IL‐6, CRP (P < .000), CT score (P < .000), or who needed nutrition support  or with electrolyte imbalance  than the optimal threshold had a worse outcome, with all‐cause death or received mechanical ventilation. Whereas, lymphocyte count  acted as a protective factor, showed that patients with higher lymphocytes had a better outcome.The median follow‐up time was 33 days, a minimum of 1 day, and a maximum of 39 days. Kaplan‐Meier analysis showed that the mean survivals of critical and non‐critical patients were 19.21 ± 2.30 and 38.60 ± 0.39, with a significant statistical difference  had a higher risk for outcome after adjustment for other cofounders. Some explanations are posited to explain the lymphopenia caused by SARS‐CoV‐2. First, the coronavirus can directly infect T cells but fail to replicate within the cells. Second, inhibitory cytokines are released by macrophages or lung epithelial cells that cause T‐cell apoptosis or prevent their proliferation.Dysregulation of immune response plays an important role in patients with COVID‐19. Results of epidemiological studies are suggestive of lymphopenia and hypercytokinemia, which may act as hallmarks of COVID‐19.COVID‐19 associates states of both immunodeficiency and hyperinflammation. The overly exuberant inflammation situations will eventually lead to critical conditions, which termed “cytokine storm”.In addition, we attempted to explore the relationship between CT score and critically patients. By scoring based on infiltration and consolidation imaging, we found that the lung injury of critical patients was more severe than that of non‐critically patients. Patients with scores three or above had a worse outcome.Furthermore, considering the effects of nutritional status and homeostasis on the disease, we evaluated the patient who needed nutrition support and corrected electrolyte imbalance via intravenous injection ≥3 days. Another study confirmed that screening for patients with COVID‐19 who are at nutritional risk, as well as in need of additional nutritional intervention, is associated with their outcomes.This study has some limitations, including the number of cases was small and clinical data were limited. Some relevant data such as PT, APTT, D‐dimer were not complete and unable to be included in the risk factor analysis.5In conclusion, the lower lymphocyte count and the higher levels of NLR, PLR, CRP, IL‐6, and CT score have a significant correlation with the severity of COVID‐19, which can be used as useful risk factors to discern critical patients or predict disease outcome. Moreover, nutritional risk and electrolyte imbalance can also be considered as evaluation indicators for patients with COVID‐19.The authors declared that there is no conflict of interest.Weili Wang, Zhi Xu, Jinghong Zhao, and Jingbo Zhang designed the study. Weili Wang, Xi Liu, Gang Liu, and Dongjing Xie collected and organized data the data. Weili Wang and Zhongxiu Zhao analyzed and interpreted the data. Weili Wang and Jingbo Zhang wrote this manuscript and all the authors revised it.This retrospective observational study was approved by the institutional Research Ethics Committee of the Chinese People's Liberation Army Joint Logistic Support Force.


Tuberculosis is currently considered a public health issue worldwide. Although a large number of hematological disorders related to tuberculosis have been described, immune thrombocytopenic purpura in patients with active tuberculosis is very rare but must be considered in a patient with no other evident cause. Tuberculosis is currently considered a public health issue worldwide. Although a large number of hematological disorders related to tuberculosis have been described, immune thrombocytopenic purpura in patients with active tuberculosis is very rare but must be considered in a patient with no other evident cause. Thrombocytopenia is a rare manifestation of active tuberculosis. We present the case of a 38‐year‐old man who presented with subacute respiratory symptoms and a platelet count of 5000 per microliter in the context of active tuberculosis, where no other causes for thrombocytopenia were found.Tuberculosis (TB) is currently considered a public health issue worldwide and one of the leading causes of morbidity and mortality in Latin America and Colombia.Thrombocytopenia is a hematological disorder characterized by a platelet count lower than 100,000 per microliter (mcl) and is considered severe with values below 20 000 per mcl. Immune thrombocytopenic purpura (ITP) is a cause of thrombocytopenia that occurs due to antibody‐mediated platelet destruction. It can be primary, or secondary to autoimmune, malignant, or infectious diseases such as systemic lupus erythematosus, lymphoproliferative syndromes, hepatitis C, acquired immunodeficiency virus, or TB.Although a large number of TB‐related hematological disorders have been described, anemia, leukocytosis, and pancytopenia are the most frequent. Thrombocytopenia is an unusual manifestation of active TB and is usually secondary to nonimmunological disorders, occurring with pancytopenia that develops after bone marrow granulomatous infiltration.2A 38‐year‐old man, who was deprived of his liberty at prison for several years until three months before his admission, arrived at another institution for ecchymotic lesions, productive cough, weight loss, anorexia, epistaxis, and low‐grade fever of 20‐day duration, without previous trauma. His past medical history was otherwise unremarkable, and he reported not having previous alterations in clinical and laboratory examinations whatsoever. Laboratory tests at admission were as follows: leukocytes 9,460/mL , neutrophils 82% (NR: 34‐67.9), hemoglobin 10.4 g/dL (NR: 13.7‐17.5) hematocrit 30 mg/dL (NR: 40.1‐51), erythrocyte sedimentation rate 83.3 mm/h (NR: 0‐15), platelets 5000/mL (NR: 163 000‐337 000), PT 11.8 (control: 11.3), PTT 29 (control: 30.9), Fibrinogen 509 mg/dL (NR: 150‐400), D‐dimer 13 080 μg/mL (NR: 0‐0.5), total bilirubin 0.95 (NR: 0‐1.2), lactic dehydrogenase (LDH) 206 IU/L (NR: 135 ‐ 225), alkaline phosphatase 80 IU/L (NR: 20‐140), folic acid 11.49 ng/mL (2.6‐17), and vitamin B12 357 ng/mL (NR: 197‐771). He had negative ELISA for HIV, negative hepatitis B surface antigen, and hepatitis C antibody; nonreactive RPR, direct bacilloscopy was negative; urinalysis had macroscopic hematuria; and urine culture was negative. A chest X‐ray showed a cavitating lung lesion with thick edges in the left pulmonary apex Figure . The casOn his fourth day of hospitalization, he developed hemodynamic instability, given by persistence of active bleeding: hematemesis, epistaxis, hematuria, and severe secondary anemia, requiring blood transfusion and apheresis platelets. He was referred to our institution in poor general conditions. On physical examination: blood pressure 110/70 mm Hg, heart rate 75 bpm, respiratory rate 13 bpm, oral cavity with petechial lesions, coarse crackles in both lungs, multiple petechiae, and ecchymoses spread all along the extremities. The rest of the physical examination was unremarkable. Laboratory tests showed leukocytes 11 200/μL, neutrophils 10 240/μL, lymphocytes 530/μL, hemoglobin 9.8 g/dL, hematocrit 28%, platelets 1000/μL, PT 15, INR 1.38, PTT 27.5, Fibrinogen 738, total bilirubin 0.95, ALT 12 mg/dL (NR: 0‐41), AST 28 mg/dL (NR: 0‐38), LDH 397 IU/L, IgA: 1.88 (NR: 0.7‐4.0), IgG: 33 (NR: 7‐16), and IgM: 1.17 (NR: 0.4‐2.3). HIV antibodies were negative, and viral load was undetectable. Chest X‐ray showed a thick‐walled cavitating image in the left pulmonary apex with effacement of the left costophrenic angle probably due to effusion or pleural thickening. A thoracic CT scan showed a left apical cavity with irregular borders and hydro‐aerial level, with nodules of inflammatory aspect, ipsilateral endobronchial dissemination, and left pleural effusion , ALT 64.4, AST 73.2, sodium 132 mmol/L (NR: 136‐145), potassium 4.49 mmol/L (NR: 3.5‐5.1); chloride 98 mmol/L (NR: 98‐107), magnesium 2.19 mmol/L (NR: 1.59‐2.56), and phosphorus 3.34 mmol/L (NR: 2.5‐4.5).The patient continued steroid therapy with oral prednisolone 1 mg/kg with a gradual dose decrease in the following month as well as supervised shortened TB treatment for six months, without adverse effects or recurrence of ITP.3TB is a common disease in prisons in developing countries, due to risk factors such as overcrowding, poverty, immunodeficiency due to other diseases, among others. Our patient was exposed to these for years prior to hospitalization.ITP is an autoimmune disease characterized by thrombocytopenia as the only hematological manifestation. The two main diagnostic criteria for ITP are thrombocytopenia in the context of a normal blood count, a normal smear, and the exclusion of primary conditions capable of causing thrombocytopenia. There are two forms of ITP: the infantile acute variety, mostly postviral and self‐limited; and adult ITP that tends to be chronic, more common in women between the second and fourth decades of life.In patients with TB, ITP is very rare. There are only a few cases. TB‐induced thrombocytopenia is more commonly caused by nonimmunological mechanisms and typically manifests in the context of pancytopenia that develops secondarily to granulomatous bone marrow infiltration. The association between TB and ITP is extremely rare.Mycobacterium tuberculosis.The pathophysiology of isolated thrombocytopenia in tuberculosis is not well known. A proposed hypothesis describes an immune destruction of platelets through circulating antiplatelet antibodies, which are probably produced by an abnormal stimulation of B lymphocytes by Other causes of thrombocytopenia, besides mycobacterial bone marrow infiltration, are histiophagocytosis, thrombotic thrombocytopenic purpura, disseminated intravascular coagulation, the immune type (present in this case), and the secondary to adverse effects of anti‐TB drugs, especially rifampicin and isoniazid.The diagnosis of TB in this case was initially based on clinical and radiological findings on the CT scan; subsequently, urine and BAL samples were positive for mycobacterial ZN‐staining, which allowed diagnosis confirmation.The first line of treatment for ITP includes IV steroids, IV immunoglobulin G, and anti‐D immunoglobulin. The use of high‐dose methylprednisolone is recommended for three to five days followed by oral maintenance therapy with gradual dose reduction completing four to eight weeks depending on platelet count follow‐up. Also, adding IV immunoglobulin G 0.4 g/kg/d for 5 days can achieve a greater and faster response in approximately 80% of the cases.The therapeutic response exhibited as a progressive platelet count increase with steroids and IV immunoglobulin G, along with a confirmed TB diagnosis, altogether supported the diagnosis of ITP secondary to TB. Some authors have reported on the therapeutic use of steroids plus anti‐TB treatment, which makes it very likely that this blood dyscrasia actually represents an actual unusual manifestation of TB.Clinically, it is known that steroids improve purpuric bleeding before the platelet count actually increases. The early effect is due to the decrease in vascular permeability, while the late effect involves immunoglobulin G. A blockade of Fc receptors in reticuloendothelial cells and suppression of the production of antibodies and their binding is suggested as a possible mechanism that may be the result of anti‐idiotype antibodies that bind to antiplatelet antibodies which modulate the immune response.In this case, methylprednisolone plus immunoglobulin G were administered, achieving significant improvement as seen in the increase on platelet count.In conclusion, TB continues to be a public health issue worldwide, especially among developing countries vulnerable populations. Comorbidities such as diabetes mellitus, chronic kidney disease, and immunosuppression can favor TB infection. Diagnosis and treatment may represent a challenge, due to its multiple manifestations, in this case, a rare hematological alteration in the presence of active infectious disease.None declared.All authors contributed to data analysis, drafting and revising the article, gave final approval of the version to be published, and agreed to be accountable for all aspects of the work. YAF and LFT: conceptualized and designed, reviewed the literature, wrote and corrected the manuscript, and involved in final approval of manuscript. EIM and LFS: conceptualized and designed, reviewed the literature, and involved in final approval of manuscript.This report was prepared in accordance with the ethical standards of the institutional ethics committee and with the 1964 Helsinki Declaration. We have approval letter of Ethics Committee in biomedical research IRB/EC No. 255‐2019 of the Fundación Valle del Lili to publish this manuscript.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the editor‐in‐chief of this journal.


Stipa species dominate large areas of the Eurasian zonal vegetation, but comparisons of germination requirements between Stipa species from different habitats is limited. We investigated the effects of temperature and water potential on seed germination of S. grandis, S. purpurea, and S. penicillata from habitats with low temperatures and relatively abundant rainfall (cool habitats) and S. glareosa, S. breviflora, S. gobiea, and S. bungeana from habitats with relatively high temperatures and low amount of rainfall (warm habitats). Seeds of species from cool habitats had a higher base (Tb), optimal (To), and maximum (Tc) temperature than those of species from warm habitats, except for the base temperature of S. purpurea. Response of six tested Stipa species to water potential differed among species but not between habitats. Median water potential for germination was lowest for S. bungeana, S. penicillata, and S. gobiea. There was a negative correlation between hydrotime constant (θH) and base water potential for 50% of the seeds of all species to germinate (ψb(50)). Germination time of seven Stipa species in response to temperature and water was well predicted by thermal time and hydrotime models. Results of the present study on germination of these seven species of Stipa may provide useful suggestions for grassland restoration in different habitats.Temperature and water potential are two important environmental factors influencing germination and subsequent seedling establishment. Seed germination requirements vary with species and with the environment in which the seeds are produced.  Seed germination is the most critical stage in the life cycle of plants, and its correct timing is essential for successful plant establishment . VariousTb), optimum (To), and maximum/ceiling (Tc) generally have been used to describe the range of temperatures over which seeds of a particular species can germinate (b (crop species), while species originating from cool growing areas had a very low Tb   . Trudgilrainfall .The effect of temperature on seed germination also can be modeled by thermal time  model, which is a very efficient method for predicting timing of germination under fluctuating environmental conditions. This approach can be more useful for predicting plant development stages than calendar date when temperatures for germination or other plant development stages are outside the experimental data range .θH) and base water potential (ψb). Base water potential for 50% germination [ψb(50)] is related to the ability of the embryo to overcome restraints to its growth imposed by the conditions of the embryo itself, the surrounding tissues, and the environment (ψb(50) germinate better at low median water potential than those with high ψb(50). The base water potential at which seed germination can occur differs with species  seeds of Stipa species from habitats with low temperatures would have a lower Tb, To, and Tc than those from habitats with a relatively high temperatures, and (2) seeds of Stipa species from habitats with low amounts of rainfall are more tolerant of water stress than those from habitats with relatively high amounts of rainfall.Seed germination response to temperature  and wateStipa species were used in this study. Seeds of Stipa grandis were collected from Xilingol League in Inner Mongolia; Stipa purpurea from Guinan on the Qing-Tibetan Plateau; Stipa penicillata from Guoluo on the Qing-Tibetan Plateau; Stipa breviflora, Stipa glareosa, and Stipa gobiea from Alax in Inner Mongolia; and Stipa bungeana from Huanxian on the Loess Plateau. The species differ distinctly in habitat: S. grandis, S. purpurea, and S. penicillata occur in habitats with relatively abundant rainfall and relatively low temperature, whereas S. bungeana, S. breviflora, S. glareosa, and S. gobiea are important species in habitats with relatively low rainfall and relatively high temperature. See Stipa species.Seeds of seven Seeds of each of the seven species were collected from several hundred plants in 2016, and the awns were removed by hand in the laboratory. To avoid the potential effect of dormancy on germination results, seeds were allowed to after-ripen in a paper bag at room conditions  for 6 months before germination experiments were initiated.-2 s-1, 400–700 nm) at 5, 10, 15, 20, 25, 30, 35, and 40°C. Germination (radicle protrusion) was monitored daily for 28 days.For each treatment and species, four replicates of 25 seeds were placed in 9-cm-diameter Petri dishes on two sheets of filter paper moisten with 5 ml of distilled water. Seeds were incubated in light  solutions were prepared according to 50) of each species were analyzed using GLM  analysis based on binomial distribution using SPSS 22.0 software.The effects of temperature and of water potential on germination percentage and rate (1/tTb), maximum or ceiling temperature (Tc), and optimum temperature (To) as described by A linear model  was used to estimate base temperature (tg) and temperature (T) as per Eqn. 3 ] from each monitoring time and suboptimal/supraoptimal temperatures were pooled and regressed against a function of time , actual seed water potential ψ (MPa), and base water potential ψb(g) (MPa) were calculated using the hydrotime model (The hydrotime constant me model  as belowP< 0.05) on percentage and rate (1/t50) of germination of all seven Stipa species (S. purpurea seeds germinated to a high percentage (> 90%) at 5°C to 35°C. Germination of Stipa species from the cool habitats was more tolerant to high temperature than that of species from the warm habitats. For example, germination of S. grandis, S. purpurea, and S. penicillata seeds from habitats with low temperature and high rainfall (hereafter cool habitats) was 93%, 97%, and 51% at 35°C, respectively, whereas it was 27%, 45%, 13%, and 46% for seeds of S. glareosa, S. breviflora, S. gobiea, and S. bungeana, respectively, from habitats with high temperature and low amount of rainfall (hereafter warm habitats). However, germination of Stipa species at low temperature differed with species but not habitat. Seeds of S. purpurea from a cool habitat and those of S. breviflora from a warm habitat germinated to higher percentage at 5°C than S. glareosa, S. gobiea, and S. penicillata, while no seeds of S. grandis or S. bungeana germinated at 5°C. Seeds of S. grandis, S. purpurea, and S. penicillata from cool habitats had the highest germination rate at 30°C, whereas the highest rate for S. bungeana, S. breviflora, S. glareosa, and S. gobiea from warm habitats was at 25°C.Temperature had significant effects ) for S. grandis, S. purpurea, S. penicillata, S. glareosa, S. breviflora, S. gobiea, and S. bungeana were 54.0 °C·d, 72.0 °C·d, 56.6 °C·d, 70.9 °C·d, 99.5 °C·d, 51.0 °C·d, and 84.5 °C·d, respectively. θT(50) values for these species at supraoptimal temperatures were 42.8 °C·d, 57.6 °C·d, 15.5 °C·d, 11.1 °C·d, 35.1 °C·d, 9.2 °C·d, and 68.0 °C·d, respectively. Consistent with estimation from extrapolation, species from cool habitats had a higher Tc than those from warm habitats. However, there was no difference between species from the two habitats in Tb, θT(50), and Tc  (-0.73 MPa) and S. grandis the highest ψb(50) (-0.21 MPa). The hydrotime constant (θH) differed with species. Species with low ψb(50) exhibited a high θH, i.e., 173 MPa·d for S. bungeana and 85 MPa·d for S. gobiea. However, S. grandis with a high ψb(50) exhibited a low θH, i.e., 31 MPa·d. There was a negative correlation between ψb(50) and θH across species tested. The hydrotime models described germination of the ies well Table 5.Stipa species from cool habitats had a higher base (Tb), optimal (To), and maximum (Tc) temperature than species from warm habitats, which is contrary to our first hypothesis. Further, the germination responses to water potential differed among the six species tested but not between habitats, which does not agree with our second hypothesis. There was a negative correlation between hydrotime constant (θH) and base water potential for 50% of the seeds of all the species to germinate (ψb(50)).We showed that germination of seeds of the Echinochloa crusgalli, Panicum miliaceum, and some perennial warm-season forage grasses  and S. penicillata (10.0°C) from the same cool habitat. Morever, seeds of S. purpurea collected in 2013 from alpine steppe on the Qing-Tibetan Plateau had a base temperature 0.1°C lower than that of this species in the present study  from central Asia germinated best at high temperature (20/32°C).The optimal and maximum temperatures for germination of n winter . Geneticn winter . Differeperature . S. purpulations . ConsistCaragana korshinskii (15.0% germination at 2.1 MPa) and Hedysarum scoparium (6.5% germination at 1.8 MPa) from semi-arid regions of northwest China, and Reaumuria soongorica (2.2% germination at 1.8 MPa) from arid regions have a higher germination capacity at lower water potential than seeds of Artemisia sphaerocephala (8.3% germination at 1.5 MPa) from semi-arid regions and Zygophyllum xanthoxylum (5.2% germination at 1.2 MPa) from arid regions. However, seedling establishment of C. korshinskii, H. scoparium, and R. soongorica is lower in arid regions than that of A. sphaerocephala and of Z. xanthoxylum  of the six Stipa species tested was species-specific but was not habitat-specific.Generally, species from dry habitats are more tolerant of water stress than those from wet habitat . LudewigS. bungeana (-0.73MPa) > S. gobiea (-0.61MPa) > S. breviflora (-0.19 MPa). Thus, there was no clear pattern in response to water potential in relation to habitat type, which is consistent with the results obtained by Stipa in our study may be that soil moisture during germination is similar between the warm and cool habitats, although the annual mean rainfall differed greatly between them (θH and high ψb(50) may have a rapid germination rate with no water stress but are strongly inhibited at low water potentials. For example, seeds of S. grandis and S. breviflora have high ψb(50) and low θH. In our study, there was a negative correlation between θH and ψb(50), indicating that seeds with high tolerance to water stress (low ψb(50)) need more time to germinate than those with high ψb(50). A high θH for germination may play an important role in preventing seed germination after a low precipitation event that is followed by drought, which would kill the seedlings  seeds of the seven All datasets presented in this study are included in the article/supplementary material.XH and YW conceived the topic. RZ and KL performed the experiments. RZ and DC analyzed all statistical data. XH and RZ wrote the manuscript. JB and CB revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To evaluate a new tangential map-based nomogram versus the axial map-based nomogram for ICRS in keratoconus.  A prospective case series study including 64 eyes of 64 patients who underwent ICRS implantation. Cone location was determined for each eye with two maps: the axial and the tangential. Appropriate ring selection was determined using two surgical nomograms: axial map-based and tangential map-based. Visual, refractive, and topographic outcomes were assessed before, as well as at 3, 6, and 12 months after ICRS implantation. D at 12 months after the procedure. In the tangential group, UDVA and CDVA improved from 0.09 ± 0.06 and 0.2 ± 0.1 to 0.5 ± 0.2 and 0.7 ± 0.2, respectively. MRSE substantially decreased from −4.9 ± 1D at 12 months after the procedure. In the tangential group, UDVA and CDVA improved from 0.09 ± 0.06 and 0.2 ± 0.1 to 0.5 ± 0.2 and 0.7 ± 0.2, respectively. MRSE substantially decreased from −4.9 ± 1D at 12 months after the procedure. In the tangential group, UDVA and CDVA improved from 0.09 ± 0.06 and 0.2 ± 0.1 to 0.5 ± 0.2 and 0.7 ± 0.2, respectively. MRSE substantially decreased from −4.9 ± 1P=0.01∗. Similarly, the gain in the CDVA was 0.4 and 0.15 in the tangential and axial groups, respectively, at 12 months, P=0.01∗. Similarly, the gain in the CDVA was 0.4 and 0.15 in the tangential and axial groups, respectively, at 12 months,  The cone location, and consequently the ring selection, was significantly different in the two nomograms with a “centralization tendency” in the tangential map. In the axial group, UDVA and CDVA improved from 0.12 ± 0.04 and 0.24 ± 0.08 to 0.28 ± 0.08 and 0.4 ± 0.1, respectively. Similarly, MRSE substantially decreased from −6.7 ± 3.3 to −1.2 ± 1.1 The tangential map-based nomogram attained better visual and refractive outcomes at 1 year. In addition, the cone location was significantly different between both maps with a centralization tendency in the tangential one. Keratoconus (KC) is a bilateral, progressive, noninflammatory disease of the cornea which often leads to high myopia and astigmatism which impair the acuity and quality of vision. It has an estimated prevalence of approximately 1 in 2000 and an incidence between 50 and 230 per 100,000 .Q value, and corneal topography; however, no nomogram is agreed upon [Intrastromal corneal ring segments (ICRS) enriched the armamentarium for KC, with promising results in topographic regularity and uncorrected distant visual acuity (UDVA), raising the hopes to reduce the need for keratoplasty , 6. ICRSeed upon . All of Even the current manufacturers as well as many surgeons depend on the axial map as a guide for the cone location and subsequently the appropriate ring selection and implantation. The tangential map, however, was superior in many aspects, including detection of subclinical KC and contact lens fitting in KC , 9. GiveIn this study, we aimed to compare the axial map-based versus a tangential map-based nomogram for ICRS implantation in keratoconus. The comparison was planned in terms of preoperative cone location matching, in addition to postoperative visual, refractive, and topographic outcomes.This was a prospective case series study conducted at Tiba Eye Center, Shebin Elkom, Menoufia, Egypt, during the period from April 2017 to July 2018 on patients with KC who are candidates for ICRS and meet the eligibility criteria. All study procedures were approved by the Ethical Committee of Menoufia Faculty of Medicine and were in accordance with the declaration of Helsinki.μm at the optical implantation zone, mean keratometry between 45 and 60D, contact lens intolerance, an uncorrected distant visual acuity (UDVA) not better than 0.5 decimal, and no visual dysfunctions other than keratoconus. Pregnant or breast-feeding mothers and patients with other ocular or systemic pathologies were excluded.We included patients with clinical keratoconus, diagnosed with pentacam , with clear central cornea, age between 21 and 49 years, minimum corneal thickness of 400 D in sphere and cylinder between before surgery and after surgery (effect size). The result of the sample size calculation was 25 eyes per group.The sample size was calculated using Power Analysis Sample Size software  setting the type 1 error (a) at 0.05 ) and the power (1-b) at 0.8. The sample size was calculated to detect a difference of at least 0.25μm for SE of more than −6.00D and less than −6.00D, respectively. For peripheral cones, Keraring segments were implanted according to the manufacturer's nomogram tables as shown in The cone location was labeled as central, paracentral, or peripheral according to the extent of the cone area. A circle that encompasses the 2 warmest colors was delineated, and its diameter was measured. The cone was central or paracentral if more than half of the diameter fell within the central 3 or 5 mm, respectively. Otherwise, it was labeled as a peripheral one. The cone location was determined for each case with both axial and tangential maps and compared for matching. For each one, the same surgical nomogram was applied respecting the cone location as determined by the map used. For central and paracentral keratoconus, 320° ICRS Keraring  was applied with the thickness of 300 and 250 A, B, or C) was conducted to emphasize the discrepancy, if any, between both maps as a guide for ICRS implantation.An alternative scenario analysis was planned. This is to determine the cone asymmetry classification type, as reported in Keraring manufacturer's nomogram, by both maps. Cone asymmetry classification was determined for each eye as proposed by the manufacturer in relation to the reference meridian  using the axial map and then matched to that using the tangential map. Two independent authors visually assessed the cone asymmetry in both maps and discrepancies were resolved by discussion or by the senior author. After that, matching the cone pattern and the subsequent nomogram  and corrected distance visual acuity (CDVA) in decimal units, manifest and cycloplegic refraction, slit lamp examination including applanation tonometry, fundus examination, and corneal topography with a rotating Scheimpflug device . The primary outcome was CDVA at 12 months after the operation. Secondary outcomes were UDVA, CDVA, manifest refraction SE (MRSE), Aiming at a standard procedure, ICRS implantation was carried out by the same surgeon (A. S) under local anesthesia using a femtosecond laser . The stromal tunnel was created at approximately 70% of corneal depth. The pockets were opened using a blunt sinskey, and the rings were implanted. Soft bandage contact lens was placed immediately at the end of surgery in all cases, and patients were instructed to avoid eye rubbing. All patients received the same postoperative treatment of topical antibiotic and steroid (Fortymox® q.i.d and Predforte® q.i.d).t-test was used to compare the means of the outcomes between both groups. The paired t-test was used to compare preoperative and postoperative values of UDVA, CDVA, SE, Kmax, Kmean, and Q value. The mean change in the above variables was analyzed using Student's t-test. Chi-squared test was used to compare categorical variables. The P value <0.05 was considered statistically significant in all these tests.Data analysis was conducted using SPSS v.24 software . Continuous variables are presented as mean ± SD. Normal distribution of the data was checked by the Kolmogorov–Smirnov test. Student's A  included 36 eyes, of which 24 were males and 12 females, while group B  included 28 eyes, of which 12 were males and 16 females. Mean age in both groups were 27.6 ± 3.8 and 23.7 ± 3.5 years for the axial and the tangential groups, respectively. Sixty-four eyes of 64 patients, 36 males and 28 females, were included in the study. They were divided into two groups according to the nomogram applied, and in other words, the map used to locate the cone: group P=0.01). Moreover, the peripheral group showed the greatest discrepancy where 26 out of 42 (61.9%) were labeled as paracentral on the tangential map although displayed as peripheral on the axial map. P=0.01∗).For the cone location, labelling the axial map as the reference one, the matching rate between both maps was 67.6%, 62.5%, and 38.1% for central, paracentral, and peripheral cones, respectively, which was statistically significant (P=0.01∗) as shown in 2For the cone symmetry classification proposed by Kera manufacturer's nomogram , a signiKmax, and Km in both groups at 3, 6, and 12 months postoperatively. As well, the Q value significantly changed from −1.28 preoperatively to −0.4 at 12 months (P=0.001) and from −1.38 preoperatively to −0.38 ± 0.5 at 12 months (P=0.003) in the axial and the tangential groups, respectively. In addition, a significant increase in the thinnest location thickness was documented for both groups at all follow-up visits. Km or Kmax with any of the visual or refractive outcomes.In both groups, UDVA and CDVA significantly improved at 3, 6, and 12 months after the surgery. Similarly, a significant reduction was noted in MRSE, P=0.02). In addition, at 6 and 12 months, the tangential group showed significantly better gain in UDVA  and CDVA . MRSE reduction was also higher in the tangential group . Tables The value of change from the baseline was calculated and compared between both groups. At 3 months, the tangential group showed significantly better gain in UDVA (0.2 ± 0.09) versus (0.11 ± 0.07) in the axial group  have been proposed and investigated as an additive surgical procedure for keratoconus correction  aiming aKeraring proposed its nomogram in 2009, relying on cone asymmetry in relation to the reference meridian. As well, many other designs were proposed and widely used: 355°, 340°, and 320°. Most surgical nomograms rely upon the axial map to determine the cone location or asymmetry. Instead of constructing a validated and reliable nomogram, simple unexplained instructions are provided with each ring –16. Few Axial and tangential maps do differ in many aspects, and a lot of studies affirm this. In apical corneal power, which is sensitive to minor changes in radius, the tangential map was superior to the axial one in local radius of curvature measurement, consequently, superior in apical power assessment. That is why tangential map was recommended for apical clearance method of contact lens (CL) fitting in keratoconus . The tanK was flattened by a mean of 2.7D and 0.9D in the central and peripheral cone groups, respectively. This was attributed to a nonhomogenous effect of CXL  and inherent corneal biomechanics. Both factors could be encountered with ICRS [The impact of the cone location on the outcomes after corneal cross-linking and corneal rings is a matter of debate. In 2012, Greenstein investigated how preoperative cone location can predict and/or influence the 1-year outcomes of CXL in progressive keratoconus and post-LASIK ectasia. In keratoconus, obtained flattening effects changed with different cone locations. Maximum ith ICRS .In the present study, 64 eyes of 64 patients were included. We aimed to evaluate if the cone location substantially differs between the axial and tangential maps. In addition, we aimed to investigate potential effects of any discrepancy on visual and topographic outcomes after ICRS implantation. While we assessed the visual and topographic outcomes at 3, 6, and 12 months postoperatively, we will emphasize the 1-year outcomes in the discussion section.In our study, the mean age was 27.6 and 23.7 years in the axial and the tangential groups, respectively. While there was no statistically significant difference between both groups, the age may seem relatively high compared to the keratoconic population. The effects of aging on keratoconic corneas are the areas of debate. For normal population, corneal stiffness increases with age due to changes in the stromal collagen. This led to the assumption that the same could happen with KC which can stabilize or, at least in part, slow the disease progression . Millodok readings. Nonetheless, the retrospective nature of the study along with the different techniques , and the nomograms applied may question the reliability of this correlation. One more point to highlight is restricting the inclusion to those with stable refraction before the procedure, an additional limitation for the obtained correlation [To extend the latter point, the potential correlation between ICRS outcomes and age was explored. An outstanding hurdle in many ICRS studies, including ours, is the narrow age ranges included. Torquetti et al.'s study was among the early studies to report regression of ICRS outcomes in progressive keratoconus in young patients , 27. Refrelation .The cone location, as previously defined, was matched only in 67.6%, 62.5%, and 38.1% for central, paracentral, and peripheral locations, respectively. Such discrepancy in matching cone locations might be attributed to the validity of the cone definition applied rather than a true difference between both maps. To compensate for this, we planned for an alternative scenario analysis. Given the fact that the manufacturer's nomograms were the most widely applied, what would have been the case if the tangential map replaced the axial one in these nomograms? The cone asymmetry classification proposed by the manufacturer is a visual interpretation of the warm areas on the map. The matching rate between both maps was highest in nomogram C, the 50 : 50 cones. Nonetheless, this “highest” matching rate was 25%, a considerably low rate, affirming inherent differences between both maps.It is not clear, after two decades of practice, how the cone location impacts, if any, the ICRS outcomes. Scanning manufacturers' nomograms reveal how centrality and symmetry of the cone could alter the ring choice; however, there was no evidence to support. To get over this, some studies restricted their inclusion to only one cone location. Cueto et al. reported the outcomes of one or two Ferrara ring segments in central KC with regular astigmatism , while LFeaturing the crucial role of cone location, some other studies evaluated the outcomes in paracentral KC. A longitudinal follow-up study reported significantly improved UDVA and CDVA 6 months after Ferrara ring implantation which remained stable for five years , 32. In D at 12 months after the procedure. In the tangential group, UDVA and CDVA improved from 0.09 ± 0.06 and 0.2 ± 0.1 to 0.5 ± 0.2 and 0.7 ± 0.2, respectively. MRSE substantially decreased from −4.9 ± 1D to −1.00 ± 1.6D at 12 months after the procedure. The tangential map-guided group had statistically significant better UDVA and CDVA, either in terms of absolute values or change values (Δ). While for MRSE, the change values (Δ), not the absolute ones, were significantly lower in the tangential group.In our study, both the axial- and the tangential map-guided nomograms showed significant improvement in terms of UDVA, CDVA, and MRSE. In the axial group, UDVA and CDVA improved from 0.12 ± 0.04 and 0.24 ± 0.08 to 0.28 ± 0.08 and 0.4 ± 0.1, respectively. Similarly, MRSE substantially decreased from −6.7 ± 3.3 to −1.2 ± 1.1In a recent study, 3 types of rings were compared: 2 symmetric 160° ring segments, 320° rings and MyoRing. The authors reported their own nomograms for central cones defined on posterior elevation maps. MyoRing and 320° rings achieved better visual and topographic outcomes than symmetric ring segments. Trying to reflect this in our study, 22 out of 28 eyes (78.6%) in the tangential group compared to 12 out of 36 (33.3%) in the axial group received the 320° rings. This can explain the better visual outcomes obtained in the tangential group .The better outcomes in the tangential group could be attributed to the higher proportion of the central/paracentral cones with more 320° rings implanted. Literature review for prior 320° segments' studies yielded only four reports which will be highlighted chronologically. The first study was conducted in Egypt by Israel and her colleagues who reported the outcomes for 4 types of rings: 160, 210, 320, and 355 . RecentlD [D at 6 months follow-up [Visual outcomes in our study are comparable or superior to all prior studies. Israel reported improved UCVA and CDVA from 0.02 to 0.06 to 0.16 and 0.32 respectively, and a reduction of spherical error from −9.5 to −0.5D . Yousif ollow-up .D. However, one limitation to consider is using two techniques for ring implantation: manual and femto-assisted. Authors did not report a subgroup analysis for the technique which may confound the obtained outcomes [Not far from this, Rocha and his colleagues reported improved UCVA and CDVA from 1.36 and 0.51 (LogMAR) to 0.63 and 0.18 (LogMAR), respectively, after 6 months of 320 rings' implantation . IncludiKmax was significantly reduced from 53.6 ± 2.5D preoperatively to 48.8 ± 3.9D at 12 months, while in the tangential group, it was reduced from 54 ± 6.4D preoperatively to 50 ± 4.7D at 12 months. Similarly, average K reading (Km) showed significant reduction in both groups. In the axial group, it was significantly reduced from 51.7 ± 5.8D preoperatively to 45 ± 3.7D 12 months, while in the tangential group, it was reduced from 51.09 ± 1.4D preoperatively to 44.5 ± 2.7D at 12 months postoperatively.In the axial group, Kmax and Km) and significantly enhanced both the UDVA and CDVA as well as improving the corneal topography [D in Km [This is in agreement with El-Raggal, who reported a statistically significant reduction in the mean keratometric reading from the preoperative values . Similarpography . Also, I[D in Km .It is not surprising to obtain better outcomes without providing an explanation for this. Such conflicting ambiguous reports, along with ours, raise many questions such as how do corneal rings really work and what does really count with them? We are not in the position to declare a nomogram to be followed by all surgeons. However, the discrepancy in the outcomes we present are too significant to discard. Considering the posterior corneal surface and high-order aberrations in further studies with larger sample size and long-term follow-up may elucidate the outcomes' influencer. Reaching a consensus for the terminology, including the cone location, and the nomograms for ICRS implantation is essential for a reproducible practice.Both axial- and tangential map-based nomograms for ICRS significantly enhanced the visual and the topographic outcomes in keratoconus, with a “centralization tendency” for cone location by the tangential map. In addition, the tangential map-based nomogram yielded better visual and refractive outcomes especially in the peripheral cones. Larger studies to validate the tangential map-based nomogram along with other available nomograms are essential for a consensus in ICRS practice.