Lycium europaeum methanol extract (LEM) on liver and kidney injuries induced by cisplatin. The phytochemical composition, the antioxidant activity, and hepatorenal injury biomarkers were investigated. Results revealed that LEM exhibited a significant antioxidant activity in vitro on DPPH radical and H2O2 scavenging assays. In the animal studies, treatment with LEM significantly reduced the effects of cisplatin intoxication on serum liver biomarkers and serum renal biomarkers. Meanwhile, LEM diminishes significantly the effect of cisplatin on the level of lipid peroxidation in liver and kidney tissues. The activities of the antioxidant enzymes  were increased in groups pretreated with LEM and quercetin. Additionally, the normal histological structures of the liver and kidney were restored after treatment with LEM. This work clearly demonstrated that L. europaeum may be useful as a drug with hepato-nephroprotective potentials.This study was designed to assess the protective effects of  Lycium, a member of the Solanaceae family, consists of 70 species distributed throughout temperate and subtropical areas. Lycium was cited in the traditional medicine of many countries. In Tunisia, the dry fruits from L. intricatum are traditionally used to protect from eye diseases [L. chinense and L. barbarum are used for longevity, infertility, and headache [Lycium shawii are used as antidiabetic [Lycium is a rich source of phenolic acids, carotenoids, tannins, flavonoids, mineral, and vitamin [L. europaeum is a shrub spread in many Mediterranean regions [L. europaeum have been conducted on the fruits. Findings obtained from 80% ethanol extract demonstrated the cytoprotective effects against human lung carcinoma and rat adrenal medulla cancer cells [L. europaeum are characterized by high content of flavonoids, anthocyanins, carotenoids, lycopene, and tannin [L. europaeum leaf extracts have antinociceptive and protective effects against CCl4-induced liver and kidney injuries in mice [L. europaeum methanol extract (LEM) was evaluated. The antioxidant activity in vitro and the hepato-nephroprotective effects of this plant were also investigated.diseases . In Chinheadache . The flodiabetic  and leavdiabetic , 5, hepadiabetic , and antdiabetic , 9. More vitamin . L. euro regions , 12. In er cells . Such acer cells , 14. In d tannin ; these c in mice . In this2O2), 2-thiobarbituric acid, trichloroacetic acid, catalase (CAT), malondialdehyde (MDA), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma–Aldrich Co. . Standards (iron (Fe), zinc (Zn), calcium (Ca), potassium (K), magnesium (Mg), manganese (Mn), cuivre (Cu), and sodium (Na)) were obtained from Merck . Superoxide dismutase (SOD) and glutathione peroxidase (GPx) assay kits were purchased from Randox Laboratory Ltd. Methanol was purchased from PanReac . All other chemicals and solvents were of analytical grade and were obtained from Sigma–Aldrich .Cisplatin (cis-dichlorodiammine platinum (II)), quercetin, butylated hydroxytoluene (BHT), dimethyl sulfoxide (DMSO), hydrogen peroxide , southwestern Tunisia, in January 2016 and deposited at the herbarium in the Faculty of Sciences, University of Gafsa, Tunisia. Leaves (450 g) were washed with distilled water and dried at room temperature (approximately 20% relative humidity and 24°C temperature) for 48 h. Then the samples were ground to fine powder using a blender  and extracted for 24 h with methanol for three times at room temperature with magnetic stirring. After filtration using Whatman number 1 filter paper, the solvent was entirely evaporated under vacuum at 40°C using a Büchi rotary evaporator, to obtain the dry extracts (LEM). The phytochemical composition of LEM included caffeic acid, gallic acid, catechin, epicatechin, apigenin, coumaric acid, luteolin, naringenin, vanillic acid, rutin, quercetin, and kaempferol [The leaves of empferol .Minerals were evaluated according to the method described by Kachiguma et al.  with minLipid-soluble pigments were estimated using the method of Arnon . A driedμg/g of dry weight:The chlorophyll contents were calculated through the following formulas and then expressed as L. europaeum extract on scavenging H2O2 was determined based on the protocol described by Ruch et al. [2O2 solution  was added to 1 mL of LEM with a different concentration . The tubes were vortexed and incubated at room temperature for 10 min. BHT was used as standards for comparison. The absorbance was measured at 230 nm. The effect of h et al.  with somμg/mL) were prepared to obtain a final volume of 1 mL and were mixed with 2 mL of a freshly prepared DPPH solution . After 30 min of incubation in the dark, the absorbance was measured at 517 nm on a UV-VIS spectrophotometer. IC50 value (amount of sample providing 50% of scavenging on DPPH) was determined from the linear regression equation obtained from the concentrations of the sample and the percentage of inhibition.The effect of the extract on DPPH radical was determined following the method reported by Bounatirou et al. . Adequatad libitum and were cared according to the Tunisian code of practice for the Care and Use of Animals for Scientific Purposes and the European convention for the protection of vertebrate animals used for experimental and other scientific purposes .Male Swiss mice, approximately 20–24 g body weight (bw), with age of about 7 weeks were obtained from the Central Pharmacy (Tunisia) and were maintained for a two-week adaptation period. Animals were fed with 15% protein food pellets  and had tap water n = 30) were divided into five groups, with six mice in each group. Hepatorenal toxicity in mice was induced by intraperitoneal injection of cisplatin dissolved in corn oil at the dose of 13 mg per kg body weight (bw) [Control. Corn oil was administered orally (p.o.) for 10 days.Group (I)LEM, 150 mg/kg bw [Group (II)Cisplatin. Corn oil was administered orally for 10 days and a single intraperitoneal (i.p.) dose of cisplatin  was administered on the 5th day.Group (III)LEM + cisplatin. Mice were treated with LEM (150 mg per kg bw doses p.o.) orally for 10 days and a single dose of cisplatin  dissolved in corn oil was administered on the 5th day, 1 h after the LEM dose.Group (IV)Quercetin + cisplatin. Animals were treated with quercetin dissolved in DMSO (0.5% v/v) at 50 mg per kg bw doses p.o. [Group (V)ses p.o.  orally fThe animals (ght (bw) . TreatmeAll animals were killed by decapitation 24 h after the last treatment. Blood samples were collected into heparinized or EDTA tubes by putting a funnel under the neck's mice after cervical decapitation to avoid stress. Blood was centrifuged at 1500 rpm for 10 min in order to obtain serum for biochemical analysis. The kidneys and livers were immediately removed and weighed for the organ weight ratio calculation.Serum liver biomarkers , aspartate aminotransferase (AST), and lactate dehydrogenase (LDH)) were assayed using commercial reagent kits from Biomaghreb (Tunisia). Biochemical markers of kidney injury ) were estimated using commercial reagent kits (Sigma–Aldrich) according to the manufacturer's protocol.About 1 g of each organ was cut into small pieces and immersed into a 2 mL ice-cold lyses buffer  using Ultra-Turrax homogenizer for 15 min and then filtered and centrifuged . Supernatants were stored at −80°C until use.Protein contents in homogenates were measured using the Folin reaction through the method of Lowry et al.  using boμL of each homogenate was added to 100 μL of trichloroacetic acid  and the mixture was centrifuged at 4000 × g for 10 min. After that, 100 μL of the supernatant and 200 μL of thiobarbituric acid reagent  were incubated for 15 min on a boiling water bath. The level of lipid peroxidation was measured as thiobarbituric acid reactive substances (TBARS) and was expressed as malondialdehyde (MDA) formation, in accordance with the method of Draper and Hadley [To estimate the degree of lipid peroxidation, 100 d Hadley  and exprμmoles H2O2 destroyed/min/mg protein. Glutathione peroxidase (GPx) activity was determined using the protocol described by Flohe and Gunzler [Superoxide dismutase (SOD) activity was estimated according to the method of Durak et al.  and was  Gunzler  and was Liver and kidney sections were fixed using formalin solution (10%). Then tissues were washed in 70% alcohol, dehydrated through a series of ascending ethanol percentage washes until 100% ethanol was reached, cleared in xylene, infiltrated with soft paraffin (melting point = 60°C), and finally embedded into molten paraffin. The paraffin sections were dewaxed in xylene and then rehydrated through a series of descending ethanol percentage washes finally embedded into molten paraffin wax. The resulting paraffin blocks were cut at 4–6 mm thickness using a rotatory microtome stained with hematoxylin and eosin and observed under a microscope.p < 0.05.Statistical analysis was performed using the SPSS version 18.0 software. All data were analyzed using ANOVA followed by the Tukey test. All values are given as mean values and standard deviation. Differences were considered significant at Minerals have significant roles in many activities in the human body. For example, the dietary potassium and calcium supplementation play an important role in the regulation of blood pressure , 28.Lycium and the information about the mineral contents are limited. The micromineral  and macromineral  profiles of L. europaeum are detailed in L. chinense and L. barbarum. Moreover, the results showed that the leaf contains a higher Fe level compared to the other micronutrients. All over, the mineral composition of Lycium is greater than or equal to other leafy vegetables [The nutritional composition of the leaf from getables .L. europaeum, μg/g dry weight), followed by β-carotene (68.02 μg/g dry weight), and lycopene (40.38 μg/g dry weight). Lipid-soluble pigments like chlorophylls, β-carotene, and lycopene, play an important in the human health [β-Carotene and chlorophyll have been known to have antioxidant properties, being capable of inhibiting lipid oxidation [β-carotene, and lycopene, makes the leaf from L. europaeum a special component for human nutrition.With regard to the pigment contents in the extracts from the leaves of n health . β-Carotxidation . They shxidation . The preL. europaeum methanol extract to scavenge the DPPH and H2O2 radicals  [L. europaeum fruit oil in the DPPH and ABTS scavenging tests. Moreover, our recent research has found that the leaves of L. europaeum consisting of bioactive compounds, including caffeic acid gallic acid, naringenin, and another 9 compounds, could protect against CCl4-induced liver and kidney injuries in mice, in vivo, according to HPLC-DAD analyses [The antioxidant assays have revealed the ability of radicals . The EC5 tannin) . Recentl tannin)  have demanalyses .Cisplatin is a chemotherapeutic drug used to treat several tumors. Unfortunately, numerous severe side effects of this drug have been reported like neurotoxicity, hepatotoxicity, and nephrotoxicity . In receL. europaeum, quercetin, and cisplatin on the body weight as well as the relative kidney and liver weights were illustrated in L. europaeum was added to the treated animals, the adverse effects of cisplatin on these parameters were reversed.The effects of p < 0.001) with cisplatin compared to the control group  in the levels of SOD, CAT, and GSH both in the liver and in the kidney as compared to control groups. These results are in accordance with earlier findings of Kumar et al. [p < 0.001). The preventive effect of LEM was similar to that of quercetin-treated mice (50 mg/kg BW). Clearly, our results indicate that the methanol extract from L. europaeum prevents the exhaustion in enzymatic antioxidants and protect from cisplatin-induced oxidative damage.The antioxidant system of defense includes three enzymes  which play an important role in preventing cells against reactive oxygen species and their harmful effects. However, prolonged use of chemotherapeutic drugs like cisplatin induced acute liver and kidney injuries, which make them more sensitive to oxidative damage . The effr et al.  who repoMDA, the final product of lipid peroxidation, is generated under overproduction of free radicals and is regarded as a marker of cell injury and cytotoxicity .p < 0.001). This increase in MDA levels following cisplatin exposure may be attributed to the overproduction of ROS, which could be associated with hepatocyte enzyme leakage and the inhibition of mitochondrial enzyme activity [p < 0.01). Conversely, pretreatment with LEM prevented the increase of MDA content in liver and kidney tissues. Meanwhile, the treatment with quercetin showed a good protective effect compared to the cisplatin-treated group (p < 0.01).MDA levels in the liver and kidney are illustrated in activity . Kidney L. europaeum is via antiperoxidative properties and potential role in defense against free radicals.One of the possible mechanisms of hepatic and renal protective actions of L. europaeum has a certain protective effect against cisplatin intoxication which is consistent with antioxidant enzyme and biochemical results.The hepatorenal protective potentials of LEM and quercetin were confirmed by histopathological examination . The conLycium europaeum. Results indicated that LEM is rich in bioactive compounds. LEM was found to have strong hepatorenal protective effect against cisplatin-induced liver and kidney injury in mice. This beneficial effect may be related to the strong antioxidant activity and free radical-scavenging capacity of this plant. Our results proved that L. europaeum is a novel source of natural antioxidant and encourage its use to prevent liver and kidney dysfunctions.The data from this study revealed, for the first time, the antioxidant activity and phytochemical content of the methanol extract from


Context: The plethora of ethnomedicinal applications of Tamarindus indica Linn. (Leguminosae), tamarind, includes treatment of human and livestock ailments; preparations are recognized antipyretics in fevers, laxatives and carminatives. African folklore has various applications of tamarind. However, in Nyasaland, domestic fowl are fed with preparations for prophylactic properties.Objectives: The objective of this study is to evaluate the antiviral properties of T. indica extract.Materials and methods:Tamarindus indica stem bark was extracted through ethanol maceration over 24 h, and the crude extract was fractionated by gravity-propelled column chromatography. Newcastle disease virus (NDV) inhibitory activity of extract and fractions were evaluated in vivo using 10-d-old embryonated chicken egg (ECE) as the medium for virus cultivation and antivirus assay. About 240 ECE were grouped into eight  and, 200 μL of the extract and fractions respectively inoculated into NDV pre-infected eggs and incubated at 37 °C. Allantoic fluid was harvested 5 d post-virus infection and assayed for haemagglutination (HA).Results: Anti-NDV assessment showed 62.5 mg/mL of crude extract and fractions: TiA, TiC and TiD to yield a HA titre of 1:128 each, while TiB showed 1:64 HA titre. At 125 mg/mL, a titre of 1:16 was recorded against TiB and TiD and, 1:8 against TiA. Similarly, crude extract and TiC, each recorded 1:4 HA titre. However, the minimum concentrations of extract and fraction for virus inactivation were 0.24 mg/mL and 0.49 mg/mL, respectively.Conclusion: The antiviral activity shown by T. indica portends novel antiviral drugs and, perhaps, as scaffold for new drugs. Tamarindus indica Linn. (Leguminosae), referred to as ‘Tamarind’, was evaluated for antiviral properties following cultural applications in Nyasaland known today as Malawi  and alcoholic intoxication. In the Asian sub-continent, the fruit is prescribed to counteract the ill effects of overdoses of Hydnocarpus anthelmintica Pierre (Flacourtiaceae) given in leprosy. Restoration of sensation in cases of paralysis has also been ascribed to T. indica pulp. In addition, ointment made of tamarind pulp has been reported to be used in purging domestic animals of vermin , was deposited at the Herbarium of the Department of Botany, University of Nigeria, Nsukka. The fleshy part of the stem bark was used in the experiment.The pulverized dried stem back (50 g) was extracted by cold maceration in 400 mL of absolute ethanol (BDH) for 24 h. The mixture was filtered through a Whatman No. 1 filter paper, and the filtrate was concentrated by the evaporation to dryness in a steady air current. Subsequently, extracts were exposed to UV rays for about 18 h for sterility and thereafter, extracts were stored in sterile containers at room temperature.Crude extract was fractionated using a glass column of an internal diameter of 8 cm and a length of 100 cm . Column grade silica gel (120–200 mesh size) was wet-packed, such that 2/3 height of the column was attained, using benzene and ethyl acetate (6:4) solvent system pre-determined with thin layer chromatography . Crude extract (20 g) was dissolved in absolute ethanol (10 mL) and, then made into paste by mixing with silica gel (5 g). The slurry was placed into the column and eluted continuously with the mobile phase (benzene/ethylacetate 6:4) under gravity. Aliquots of 20 mL were collected and monitored by bands formed and distance travelled on TLC. Fractions with similar mobility on TLC were pooled and the four total distinct fractions were designated as TiA, TiB, TiC and TiD. The solvent in the fractions was evaporated under steady air current at room temperature.Velogenic Newcastle disease virus strain (KVD-NDV) was obtained from Nigerian Veterinary Research Institute (NVRI) Vom, Plateau State, Nigeria. The freeze-dried virus was re-suspended and activated in accordance with the methods of Nwodo et al. .T. indica stem bark was inoculated and the eggs were incubated at 37 °C in a humidified incubator. The eggs were candled daily to observe embryo viability. Eggs with dead embryo within 24 h post-inoculation were discarded but the allantoic fluid of eggs with dead embryo 24 h post-inoculation were harvested following 4 h chilling at +4 °C and spot-checked for haemagglutination (HA). Eggs with viable embryo at 120 h were likewise chilled at +4 °C, the allantoic fluid was harvested and spot-checked for HA. Allantoic fluid harvests were pooled into sterile sample bottles, chilled at −20 °C and stored until further use. A total of 240 embryonated chicken eggs were used for the study. The eggs were grouped into eight thus, representing three controls, four fractions and the crude extract. Each group had about 30 eggs which were sub-grouped into six with each sub-group having five eggs in accordance with the treatment levels (extract and fraction concentration).Ten-day-old embryonated chicken eggs (five replicates) were inoculated with 200 μL of four hemagglutinating unit (HAU) test virus strains (≈ 1:128 virus titre) in accordance with the standards of the organization international des epizooties [OIE] (2004). Subsequently, 200 μL of 250 mg/mL of the crude ethanol extract of T. indica antiviral assay, about 200 μL of three different concentrations  of the crude extract and four chromatographic fractions  were inoculated into 10-d-old embryonated chicken egg. Similarly, the minimum concentrations of the T. indica extract and fractions with inti-NDV activity were quantitatively assessed. Virus and extract (crude and fractions) inoculations were through the allantoic and the chorioallantioc routes. The vents on the eggs were sealed with Wax-Vaseline mixture and re-incubated in a humidified incubator in the same condition as stated earlier. Allantoic fluid harvest and storage were as previously described.In addition to the 100).All allantoic fluid harvests were tested for HA using the micro-titration method OIE . Two-folT. indica, stem bark, crude ethanol extract and column chromatographic fractions evaluated on velogenic NDV (KVD-NDV) showed positive activity. The allantoic fluid harvests treated with 62.50 mg/mL of the crude ethanol extract of the stem bark and fractions; TiA, TiB, TiC and TiD, each showed a positive spot HA while at a higher concentration of 125 mg/mL, only fractions TiA, TiB and TiD showed positive HA  with potent antiviral activity separated into fraction TiC. To buttress this point, when compared with other fractions TiC and the crude extract showed higher inhibition to the growth of NDV. Fraction TiA was next in terms of antiviral activity while other followed suit as was shown in the results. These observations are in consonance with the views of other works which have shown plant active chemicals including tannins, flavonoids and terpenes as possessing antiviral potentials  of T. indica may have been active against hemagglutinin and/or neuraminidase thus, accounting for the absence of HA after T. indica treatment. Perhaps other components relevant for viral replication, including enzymes, were also impaired. However, this conclusion cannot be inferred with the findings of this work as further investigation is required. Nonetheless, it will be pertinent to isolate and characterize the active antiviral principles in T. indica as to ascertain novelty, and as well add to the existing body of knowledge.The anti-NDV activity demonstrated by


We aimed to evaluate the accuracy of coronary computed tomography angiography (CCTA) with a low-radiation protocol and iterative model reconstruction (IMR), in comparison with invasive coronary angiography (ICA). Sixty-one patients  with suspected coronary artery disease who underwent CCTA and ICA were retrospectively enrolled. CCTA was performed with low tube voltage (80 or 100 kVp), low tube current (100–200 mAs), prospective ECG triggering, and IMR using a 64-slice computed tomography scanner. Coronary artery disease was defined as luminal narrowing of >50%, as assessed using CCTA and ICA. The sensitivity, specificity, positive (PPV) and negative (NPV) predictive value, and accuracy of CCTA were examined. The mean radiation dose of CCTA was 1.05 ± 0.36 mSv. No non-diagnostic segment was noted. The sensitivity, specificity, PPV, NPV, and accuracy of CCTA were 86.4%, 96.1%, 80.3%, 97.5%, and 94.6% on a per segment basis, 93.1%, 94.7%, 88.3%, 97.0%, and 94.2% on a per vessel basis, and 100%, 83.3%, 93.5%, 100%, and 95.1% on a per patient basis, respectively. In conclusion, a low-radiation CCTA protocol with IMR may be useful for diagnosing coronary artery disease, as it reduces the radiation dose while maintaining diagnostic accuracy. The diagnostic performance of prospectively ECG-triggered CCTA with lowered radiation exposure by decreasing the scanning duration showed a high negative predictive value (NPV), when using stenosis by invasive coronary angiography (ICA) as the reference in patients with low-to-intermediate probability for CAD6.Coronary computed tomographic angiography (CCTA) is a useful, non-invasive diagnostic imaging modality for the detection of coronary artery disease (CAD). Various strategies and scanning protocols enable a reduction in the radiation dose used for CCTA, including high-pitch spiral acquisition, prospective ECG triggering, tube voltage lowering, and tube current modulation8. To our knowledge, a few recent studies have evaluated the diagnostic performance and clinical feasibility of CCTA for CAD using low tube voltage and the model-based iterative reconstruction algorithm (MBIR), which is not yet commercially available because of the extended reconstruction time; these studies have showed relatively low specificity10. In contrast, IMR can be used in daily practice; however, while the good image quality of low-dose CCTA with IMR has been reported8, its diagnostic accuracy has not been well-established.Recently, low-radiation CCTA has been used to reduce radiation exposure with the low tube voltage method. However, increased noise is a problem of this technique, which can affect image quality and diagnostic performance. The newly introduced, knowledge-based iterative model reconstruction (IMR) with a fully iterative algorithm can now be clinically used to significantly reduce noise, while maintaining image quality, as compared to iterative reconstruction (IR) with tube voltage loweringHere, we aimed to assess the accuracy of CCTA using low tube voltage, the IMR technique, prospective ECG triggering, and a radiation dose of approximately 1 mSv, in comparison with ICA, in patients with suspected CAD. In particular, we evaluated the diagnostic accuracy on a per-segment, per-vessel, and per-patient basis, and determined its impact and the factors underlying misdiagnosis on CT images.In this study, we examined a total of 61 subjects . All CCTA procedures were performed without complications. The mean radiation dose of the 61 patients was 1.05 ± 0.36 mSv  were considered excellent (Grade 4), 293 segments (38.2%) were considered good (Grade 3), and 84 segments (10.9%) were considered adequate (Grade 2). None of the segments were considered non-diagnostic (Grade 1). The mean image quality was 3.40 ± 0.68. On per-segment analysis, the image quality of the RCA, LM, LAD, and LCx were found to be 3.70 ± 0.53, 3.90 ± 0.35, 3.41 ± 0.62, and 3.35 ± 0.71, respectively , whereas a reduction in the effective dose by 46% was observed in studies with scans performed at 100 kVp, as compared to scans performed at 120 kVp 14. Furthermore, in a previous study, we found that the use of 80 kVp yielded a reduction in the mean radiation dose by nearly 50% to sub-mSv levels, as compared to CCTA at 100 kVp15.However, the issue concerning the radiation dose during CCTA is important, considering the potential stochastic cancer risks associated with medical radiation exposure17. The relevant use of the lower kVp for CCTA as well as IMR can further provide reduced radiation doses without compromising image quality.IMR relies on statistical and system models, and views reconstruction as an optimization process. Newer techniques, such as the current one, may simultaneously provide further reduction in image noise, potential dose savings, and improved image quality under conditions of reduced tube voltage10.Our study with prospectively ECG-triggered CCTA at low kVp with the IMR technique showed clinically acceptable diagnostic accuracy, consistent with that in previous reports using other various reconstructive technologies for reducing radiation dose exposure. Based on our previous study, low-radiation CCTA using IMR is clinically feasible with appreciable image interpretability, and has the advantage of the executive time of reconstruction, as compared to other reports of the feasibility of submillisievert CCTA scanning with the latest-generation iterative reconstruction algorithms in clinical settings11. Previously reported CCTA with 80–100 kVp/150–210 mA scan protocol using MBIR showed 85% accuracy per vessel analysis9. Despite the reduced radiation dose (approximately 0.29 mSv), its extended reconstruction time causes difficulty in applying MBIR-CCTA in practice9.In the present study, low-radiation CCTA (1.05 ± 0.36 mSv) with low tube voltage (80 or 100 kVp) and IMR yielded high sensitivity (86.4%), specificity (96.1%), PPV (80.3%), and NPV (97.5%) in a per segment-based analysis. These results are comparable to previous findings for CCTA obtained from prospectively triggered studies using the IR algorithm with showing high diagnostic accuracy  in patients with suspected CAD20. Although low tube voltage settings can increase the blooming artifacts from calcification, under low radiation dose conditions, the use of IMR algorithms for suppressing blooming artifacts in the presence of coronary calcium can reduce the calcium scores and image noise, in comparison with the traditional filtered back projection (FBP)22. However, this factor may still represent the major cause of false positive CCTA results and image artifacts. Another factor affecting false positive results, which are misinterpreted as non-calcified plaques, is the streak artifact as a result of X-ray beam penetrability, which reduces the effective attenuation adjacent to the dense calcification. In the present study, false negativities were primarily affected by inadequate vessel attenuation at the distal small branches, as a result of the limited spatial resolution on MDCT that causes the blurring of vessel margins . The other 4 cases of false negative diagnoses were caused by eccentric calcifications on opposite sides of the vessel wall, which led to the underestimation of coronary artery stenosis severity, as reported in a previous study23.Our findings suggest that the diagnostic accuracy of CCTA for the detection of coronary artery stenosis was affected by various factors, similar to the previously reported factors for misdiagnosis on CCTA. The false positive results were primarily affected by the blooming artifact due to a greater extent of dense calcification in coronary arteries . The presence of densely calcified plaques accentuated the partial volume averaging of different densities within a single voxel, thus leading to worse coronary lumen visualization due to insufficient spatial resolution and consequently to overestimation of luminal stenosis24. This finding may be related to the fact that one diseased segment is sufficient for detection of CAD in a patient, and that this patient may have additional diseased segments undetected on CCTA. Thus, CCTA may be currently suitable for excluding CAD at the patient level, or for detection of stenoses at the segment level25.Based on the analysis of CCTA results from patients who underwent ICA, low-dose CCTA may be suitable for excluding CAD in low-to-intermediate probability populations from the favorable NPV on per-patient, per-vessel, and per-segment bases. Moreover, there was an inverse correlation between sensitivity and specificity in comparison of segment-based and patient-based analysis (86.4%/96.1% vs. 100%/83.3%), consistent with the findings of other meta-analyses27. Several reports have suggested that the contrast media dose at 80 kVp CCTA could be decreased via iodine enhancement, with acceptable image quality30. Low contrast-dose protocols could protect renal function by using a diagnostically appropriate amount of contrast medium for low-radiation CCTA. Therefore, there is a need for further investigation regarding low-radiation and low contrast-dose CCTA protocols with IMR reconstruction.With these low-radiation CCTA protocols, it is also possible to reduce the amount of contrast agent administered, because the iodine attenuation increases with a decrease in the tube voltage as a result of contrast enhancement by reducing the energy of X-ray photons, and the mean k-absorption edge of iodine is closer to the reduced voltage2) in this single-center study. Second, there might be a referral bias because patients for whom CCTA excludes a stenosis are rarely sent for follow-up angiography, which means that lesions could have been missed. However, in this retrospective observational study, we recruited patients who underwent ICA in accordance with the relevant clinical indications. Third, the effective radiation dose could be further reduced if the tube current of the patients was more strictly modulated. We used the CCTA scan protocol with 80 kVp/100–200 mAs or 100 kVp/100–150 mAs, according to the BMI of the patients. The use of the IMR techniques could reduce image noise and permit further reduction of the radiation dose, as data can be acquired using a lower tube current (<100 mAs)9. Although the use of lower tube current results in noisy images, IMR could reduce the image noise without reducing their diagnostic quality.This study has certain limitations. First, the main limitation is the small sample size, retrospective design and small body size of the study subjects  with a conversion coefficient of 0.014 mSv/(mGy × cm). There is a need for further investigation regarding use of the new cardiac-specific factors to ensure more accurate dosimetry; this can contribute to benefit-risk calculus in cardiac imaging strategies, and support optimization of radiation safety for patients.Recently, a new conversion factor for estimating effective doses during CCTA was presented, such that a cardiac-specific conversion factor of 0.026 mSv/(mGy × cm) is appropriate to estimate effective radiation doses; this reflects a new tissue-weighting factorIn conclusion, a low-radiation CCTA protocol with low tube voltage, low tube current, prospective ECG triggering, and IMR could be a useful strategy for reducing the radiation dose of CCTA, while maintaining the diagnostic accuracy. Our findings suggest that IMR enables the accurate non-invasive diagnosis of CAD with CCTA at a 1 mSv fraction of effective radiation.This study was approved by Gangnam Severance Hospital ethics committee/institutional review board (3-2016-0282) and the requirement for written informed consent was waived due to the retrospective nature of this study. All methods were performed in accordance with the relevant guidelines and regulations.The study was designed as a retrospective study. From January 2014 to February 2016, a total of 4078 patients with suspected CAD were referred to our hospital for ICA. Among these, 411 patients who underwent CCTA within 2 months prior to ICA were examined; 314 patients were excluded because they underwent CCTA at 120 kVp or because they underwent CT with retrospective ECG-gated data acquisition. Moreover, we excluded patients who underwent CCTA using reconstructive methods such as traditional FBP or hybrid IR (n = 4), or those who underwent CCTA for stent evaluation (n = 16), bypass graft evaluation (n = 15), or both (n = 1). Finally, a total of 61 patients  were enrolled in our analysis  with prospective ECG triggering and a step-and-shoot axial scanning technique. CT scanning was performed from the carina to the diaphragm in a craniocaudal direction at the end-inspiratory pause. When the pre-examination heart rate was higher than 65 beats per min, a β-blocker  was administered per os 1 hour before the examination, without additional intravenous injection.2 and 80 kVp for BMI < 25 kg/m2); detector collimation with dynamic z-focal spot imaging, 64 × 0.625 mm; gantry rotation time, 400 ms; table feed per rotation, 4 cm; and center of the imaging window, 70–80% of the R-R interval.The CT scanning parameters were as follows: tube voltage and tube current, 80 kVp and 100–200 mAs or 100 kVp and 100–150 mAs , was administered intravenously via an antecubital 18-gauge catheter at a rate of 0.50–0.75 mL/kg for 15 s, followed by the administration of 50 mL of normal saline using a power injector . Data acquisition was started 7 s after the trigger threshold of 130 HU was achieved at the proximal descending aorta using the real-time bolus tracking technique. All patients performed the breath-hold maneuver successfully. The effective radiation dose for CCTA was calculated by multiplying the dose-length product (DLP) with a conversion coefficient of 0.014 mSv/(mGy × cm).CT images were reconstructed using iterative model reconstruction . The images were sent to a picture archiving and communication system . The reconstruction parameters were as follows: slice thickness, 0.9 mm; increment, 0.45 mm; field of view, 15–23 cm; pixel image matrix, 512 × 512; and kernel, XCC. Post-processing and reconstruction were performed with multiplanar reformatted (MPR), curved planar reformatted (CPR), and maximum intensity projection (MIP) images using commercially available software .32. The segments were included for analysis only if the diameter of the origin was equal or larger than 1.5 mm.All images were reviewed and interpreted using the PACS workstation. Two radiologists (with more than 10 years of experience interpreting cardiac images) who were blinded to patient history and the results of coronary angiography evaluated the CCTA findings. Coronary arteries were divided into 18 segments according to the Society of Cardiovascular Computed Tomography guidelines2 at the ascending aorta. Image noise was evaluated in CCTA based on one standard deviation of the attenuation value at the ascending aorta. The SNR was calculated as vascular attenuation/image noise. The CNR was calculated as [(attenuation of vessel) - (attenuation of the adjacent perivascular fat)]/image noise.The vascular attenuation values were measured using a round region of interest larger than 1.5 cm23.The qualitative image quality of the 18 coronary segments was analyzed using a 4-point grading scale as follows: Grade 1 (poor/nondiagnostic), severe image degradation or discontinuation of vessel contour that prevented vessel lumen evaluation; Grade 2 (adequate), moderate image degradation that impeded vessel lumen evaluation; Grade 3 (good), minor image degradation that did not affect vessel lumen evaluation; and Grade 4 (excellent), no image degradation All reconstructed images, including axial source images and MPR, CPR, and MIP images, were analyzed to evaluate stenosis. Half of the sum of the normal proximal and distal lumen within the same segment was used as the reference for percent stenosis. Luminal narrowing greater than 50% was regarded as significant stenosis. If there was more than one lesion within the same segment, the worst stenosis percentage was used for the analysis. The results of CCTA were compared to those obtained with ICA. Subsequently, two radiologists re-evaluated the cases for false positive and false negative segments, and the possible factors affecting diagnostic accuracy, such as location, vessel wall condition, coronary calcium scores, vessel attenuation, vessel wall motion, and other possible factors, in each corresponding segment, were discussed. Discrepancies, if any, were resolved by consensus.ICA was performed with standard techniques via radial approach. All images were digitalized for offline analysis using commercially available software . Segments were included for analysis at the bifurcation area only if the diameter was equal to or larger than 1.5 mm. Using at least 2 image planes, quantitative measurements were performed as follows: degree of stenosis (%) = [/the reference diameter]*100. The reference diameter was determined as the mean of the normal proximal and distal lumen within the same segment. Each vessel was considered to have significant stenosis if the stenosis was greater than 50%.Continuous variables are expressed as mean ± standard deviation and categorical variables are expressed as frequencies or percentages. Shapiro-Wilk tests were performed to evaluate the distribution of the data Inter-reader agreement for the image quality assessment of CCTA was evaluated by using κ statistic. A κ value of 0.00–0.20 signified none to slight agreement; 0.21–0.40, fair agreement; 0.41–0.60, moderate agreement; 0.61–0.80, good agreement; and 0.81–1.00, excellent agreement. The sensitivity, specificity, PPV, NPV, and diagnostic accuracy of low-dose CCTA were calculated by using ICA as a reference, on a per-segment, per-vessel, and per-patient basis. All statistical analyses were performed with the SPSS 20 Statistical Package for the Social Sciences .Supplementary Tables and Figure


N = 10/brand) in resin and the repetitive application of torsional stress  by an endodontic motor with autostop when the file fractured. The number of loading cycles to fracture was recorded and analyzed by Kruskal–Wallis and Mann–Whitney U tests with Bonferroni's correction. Bending resistance of the instruments was tested using a cantilever bending test to the 3 mm point from the tip (N = 10/brand). The stress was measured when deflection of 3 mm was subjected and statistically analyzed with a one-way analysis of variance and Tukey's honest significance difference test (α = 0.05). V2H withstood the highest number of load applications during torsional fatigue testing (p < 0.05), followed by NRT, PTN, and OS, where the differences between NRT and PTN (p=0.035) and between PTN and OS (p=0.143) were not statistically significant. V2H showed the lowest bending stiffness, followed by NRT, PTN, and OS (p < 0.001). Thermal treatment of NiTi wire resulted in improved mechanical properties, and controlled memory wire provided improved flexibility and torsional fatigue resistance.Nickel-titanium (NiTi) rotary files have enabled efficient root canal preparations that maintain the canal center with fewer aberrations compared to hand files. However, NiTi rotary files are susceptible to fracture, which can thereby compromise root canal treatment. Therefore, NiTi files have been developed to enhance fracture resistance by modifying design and thermal treatment. The objective of this study was to compare the torsional fatigue resistance and bending resistance of NiTi files manufactured from different alloys and treatments. ProTaper NEXT X2 , V taper 2H , NRT (heat-treated), and One Shape  instruments of tip size #25 were compared. Torsional fatigue was evaluated by embedding the 3 mm tip of each instrument ( NiTi rotary instruments have enabled easier and faster root canal preparations that maintain the canal center with fewer aberrations . HoweverNiTi alloy exists in two different temperature-dependent structures, namely, austenite (high-temperature or parent phase) and martensite phase (low-temperature phase) . MartensM-wire is a superelastic NiTi wire that contains some R-phase and martensite under clinical condition . ProTapeCM wire is manufactured by a thermomechanical process that modifies its phase transition temperature , to attaNRT  is manufactured by the thermal treatment, and according to the manufacturer, only 5 mm tip is subjected to the thermal treatment. Superior cyclic fatigue resistance of NRT has been observed when compared to other NiTi files . The manThe measurement of torsional strength in NiTi instruments has been performed with a torsiometer, according to the International Standards Organization (ISO) 3630-1. The file tip is clamped rigidly and a torsional moment applied. At the time of fracture, maximum torque and angular deflection are recorded. However, these static conditions rarely occur during torsional failures in clinical practice. Therefore, Park et al. have developed torsional fatigue testing with a torque-controlled endodontic motor to better mimic clinical conditions .The objective of this study was to compare the torsional fatigue resistance of PTN, V2H, NRT, and OS by using a torque-controlled endodontic motor that simulated their use in clinical practice. Additionally, their flexibility was compared by measuring the bending resistance of these NiTi rotary files. The null hypothesis was that there were no differences in torsional fatigue resistance or bending resistance between the PTN, V2H, NRT, and OS instruments.NiTi rotary files PTN (Dentsply Maillefer), V2H , NRT (Mani), and OS (Micro Mega) of 21 mm length and identical ISO tip size #25 were selected. NRT and OS have a constant 6% taper. PTN has a 6% taper at 3 mm from the tip , followed by a 7% taper up to 9 mm, and then the taper decreased up to 16 mm. V2H has a 6% taper at the tip area, followed by a decreasing rate of taper along the shaft. OS and V2H have convex triangular-shaped cross sections at 3 mm from the tip, PTN has a rectangular cross section, and NRT has a modified rectangular cross section at 3 mm from the tip, according to the manufacturers.N = 10/group) was evaluated according to the method described by Park et al. [U tests with Bonferroni's correction. The fracture surfaces (N = 3/group) were cleaned, sputter-coated with a conductive carbon in a sputter coating unit , and examined by scanning electron microscope .The torsional fatigue resistance of each instrument  was evaluated by a cantilever bending test, according to the previous study with a universal testing machine  [The bending resistance of each file  . Each fihttp://rsbweb.nih.gov/ij). Additionally, new instrument of each brand was examined to figure out helical angle and pitch length, and the helical angle was measured using ImageJ software.Three unused instruments in each group were embedded in a clear resin and sectioned 3 mm from the tip with an IsoMet Low Speed Saw . A SEM was used to examine the cross section, and then the cross-sectional area and inner core area at 3 mm cross sections were measured using ImageJ software (α = 0.05).The statistical difference in the bending resistance was analyzed with a one-way analysis of variance and Tukey's honest significance difference test (p < 0.001) and OS the least. When two brands of NiTi files were compared with Mann–Whitney U test, there were 6 comparisons. After adjusting Bonferroni's correction, p value lower than 0.0083 (0.05/6) was regarded as statistically significant. The differences between OS and PTN (p=0.143) and the difference between PTN and NRT (p=0.035) were not statistically significant. The NRT was significantly resistant to torsional fatigue compared with OS (p=0.002).The fractured surfaces of NRT, OS, and PTN had a topography that showed typical patterns of torsional fracture with circular abrasion marks Figures . Howeverp < 0.001).Bending resistance test result is presented in At 3 mm from the file tip, the cross sections of OS and V2H were convex triangular-shaped, whereas that of PTN was rectangular-shaped and that of NRT was teardrop-shaped . The croSeveral factors influence the torsional resistance of NiTi rotary files. These include size, taper, flute depth, pitch length, core area, cross-sectional shape, and alloy treatments , 20–24. A large number of threads are associated with shorter pitch length and greater helical angle. Prior studies reported that NiTi rotary files that have shorter pitch length and greater helical angle exhibit superior torsional resistance , 25. TheProprietary thermal treatments for the instruments were patented by the manufacturers and the details disclosed. It appears that M-wire possesses some martensite and R-phase, and NRT possesses some R-phase NiTi at room temperature , 26. HowFractographic analysis by SEM revealed circular abrasion marks and skewed dimples around the center of rotation of the fractured surfaces for OS, PTN, and NRT Figures . In contTorsional fracture occurs when the instrument tip is wedged within constricted canal space and the shaft continues to rotate . This taTo evaluate the bending properties of endodontic instruments, the ISO 3630-1 established a bending test, which involves clamping 3 mm of the tip of each instrument into a chuck and applying an angular deflection of 45° . Bendingp < 0.05), although it presented the smallest cross-sectional area among the tested NiTi files. This finding has corroborated with previous studies [The flexibility of these NiTi rotary files is depended on the properties of their alloy, rather than their cross-sectional geometry. OS, which was made from conventional NiTi alloy, exhibited the highest bending stiffness . According to Chang et al., differential scanning calorimetry of V2H showed that it was composed of a mixture of austenite and R-phase at room temperature, since the austenite finish temperature (33.25°C) is above room temperature [V2H presented the lowest bending moment (perature . The criperature . Howeverperature , so thatAlthough V2H and PTN have variable tapers, this feature did not increase their flexibility in this study. PTN instruments with variable taper were found to be less flexible than the NRT, which has a constant taper. However, additional experiments are required with NiTi rotary files that have identical alloy and cross sections to be able to adequately compare the influence of constant versus variable taper. The present study measured torsional fatigue resistance using a torque-controlled endodontic motor, to mimic clinical condition. Maximum degree of rotation cannot be obtained by this method, and further study on the torsional fracture resistance test according to ISO 3630-1 is needed.NiTi rotary instrument manufactured from CM wire by proprietary thermomechanical procedures had greater bending flexibility and torsional fatigue resistance than conventional and M-wire NiTi instruments.


We aimed to explore the correlation between ketamine abuse and lower urinary tract symptoms (LUTS) and epidemiology of ketamine cystitis. Questionnaire records of ketamine abusers, such as sex, age, and details of using ketamine, including consumption method, amount, duration of ketamine use, and LUTS, were obtained from two private rehabilitation centers. We analyzed these factors and established a severity forecasting module. One hundred and six ketamine abusers completed the questionnaires. LUTS showed an onset time of 24.67 ± 26.36 months among ketamine abusers. Overactive bladder symptom score, international prostate symptom score-storage, interstitial cystitis symptom index, interstitial cystitis problem index, and visual analogue scale score were 5.25 ± 4.43, 5.95 ± 5.72, 10.96 ± 6.66, 9.73 ± 5.82, and 2.55 ± 3.18, respectively. All symptom scores were positively correlated with the duration of ketamine abuse. Ketamine snorting was significantly correlated with all symptom scores compared to smoking. Hydrodistention, intravesical hyaluronic acid instillation, intravesical injection with botulinum toxin, and hyperbaric-oxygen therapy showed better effect than oral treatment. Ketamine can induce severe storage symptoms, such as frequency or nocturia depending on the duration of abuse. Ketamine snorting may cause worse LUTS than smoking. Combining ketamine and other substances may exacerbate LUTS. Intravesical therapy may lead to better outcomes than oral treatment. It is currently used for general anesthesia and pain management for chronic neuropathic or malignant diseases3.Ketamine is a noncompetitive N-methyl-D-aspartic acid receptor complex antagonist and phencyclidine derivative, which was first synthesized in 1962 by Calvin Lee Stevens6. Many adolescents use it because of its low price and easy usage. Long-term ketamine abuse may cause severe lower urinary tract symptoms (LUTS), which are very similar to those of interstitial cystitis/bladder pain syndrome (IC/BPS) including increased urinary frequency, urgency, nocturia, intractable dysuria, hematuria, and bladder pain. Since the first report of ketamine-associated urinary tract damage in Canada in 20077, the number of reported cases in recent years has been increasing. Although a study of LUTS in ketamine users reported a prevalence of 26.6% in non-treatment-seeking people8, epidemiological data in Taiwan are still lacking.Ketamine has become a popular recreational drug in nightclubs in Taiwan and other areas, such as Hong Kong and the UK, in the past 10 years10, there are six-line options for interstitial cystitis. However, the most important step in the management of ketamine-associated LUTS, which differs from interstitial cystitis, is the discontinuation of ketamine use.The treatment for ketamine-associated LUTS is challenging and it resembles that for interstitial cystitis. According to guidelines of the American Urological AssociationWe conducted an in-depth epidemiological study to better understand the correlation between ketamine abuse and LUTS in Taiwan. We hypothesized that various ketamine consumption parameters would be associated with LUTS.P < 0.05, Fig. P = 0.016). Combined use of ketamine and 3,4-methylenedioxy-methamphetamine (MDMA) significantly increased the ICPI score (P = 0.034).Ninety-five men and 11 women (24.87 ± 6.44 years old) submitted the questionnaires. The average age of starting ketamine use in this study was 16.68 ± 3.35 years. The duration of ketamine use was 75.45 ± 43.9 months, and cessation interval was 12.79 ± 13.47 months. Eighty-nine (84%) ketamine users developed LUTS after using ketamine for 24.67 ± 26.36 months. Ketamine use methods were snorting (34%), smoking (33%), and both (33%). Ninety-one (92.9%) subjects used ketamine more than once daily. Most of the abusers used no more than 10 gm per day . The survey showed that in the treated group, 49 (89.1%) abusers received oral medication for LUTS and 26 (53.1%) of them achieved symptom relief. Other treatments included cystoscopy with hydrodistention, intravesical hyaluronic acid instillation, intravesical injection with Botulinum toxin, and hyperbaric-oxygen therapy, which showed better effect on LUTS than on oral medication (Table Fifty-one ketamine abusers (48.1%) did not seek any treatment. All of the scores in the non-treated group were lower than those in the treated group  may exacerbate LUTS. Half of the ketamine abusers with LUTS did not seek medical advice.11 and drug misuse declaration from 2009 to 2010 in England. Previous literature has shown that the age of first-time ketamine use was approximately 16 years, which is compatible with our data12. Many abusers during this period are easily influenced by their peers.Ketamine use was found to be predominant in men. This finding is in line with a USA-based report from 2007More than 50% of ketamine abusers had LUTS such as increased urinary frequency and urgency, nocturia, urethral pain, and incomplete emptying, while increased frequency, incomplete emptying, and nocturia were the most bothersome problems of ketamine abusers.Snorting and smoking are the most frequent ways to use ketamine. Ketamine abusers may chop up ketamine crystals into powder by X-ACTO knife or razor blade, and the powder is then inhaled directly. Once inside the nose and sinuses, the drug is absorbed from the nasal mucosa quickly. The onset time for euphoria is about 3–5 minutes. Our data showed that the frequency of smoking and snorting was nearly equal; however, ketamine snorting caused more severe LUTS Fig. . This isP = 0.016). Cannabinoids are the active components of marijuana, and select cannabinoid receptors, CB1 and CB2, have been identified in the human detrusor and urothelium13. Activation of these receptors could modulate bladder afferent activity and the micturition reflex14. In patients with painful bladder syndrome and idiopathic detrusor overactivity, the density of CB1 expression is enhanced in line with afferent sprouting15. This might explain the worse storage symptoms in the abusers who combined the use of ketamine and marijuana.All the symptom scores were positively correlated with the duration of ketamine abuse, implying that more the contact time with ketamine, the more obvious LUTS and bladder pain become. It generally takes about 2 years to develop LUTS in ketamine abusers. In our study, OABSS significantly increased with the combined use of ketamine and marijuana (P = 0.034). The major mechanism of MDMA is central stimulation and a rapid liberation of noradrenaline from peripheral adrenergic terminals. Ketamine may exacerbate MDMA-induced central dopamine toxicity. Whether MDMA worsens LUTS via central stimulation is to be clarified by further research.Combined use of ketamine and MDMA significantly increased the ICPI score  for treatment-resistant depressionThe main limitation of this study is that there was only subjective assessment and a lack of objective assessment. There was also lack of control and objective assessment in the study. The results may have sex bias as the study had few female participants. Details of oral medication could not be verified; thus, we were not able to determine which medicine was effective to relieve LUTS caused by ketamine abuse.Ketamine abuse can cause voiding dysfunction. The severity of storage symptoms is correlated with the duration of ketamine use. Ketamine snorting may cause worse LUTS than smoking. The combined use of ketamine and other substances, such as marijuana and MDMA, may exacerbate LUTS. Intravesical therapy may produce better outcomes than oral medical treatment. With this model as a platform, further prospective studies are warranted to investigate the appropriate choice of treatment for this new clinical entity.The self-administered questionnaires were employed in a cross-sectional design and ethical approval by the Institutional Review Board of Tri-Service General Hospital was obtained for data collection and analysis. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study.Before answering the questionnaires, all participants were provided a verbal explanation about the contents of questionnaires and they provided informed consent. The scales included overactive bladder symptom score (OABSS), International Prostate Symptom Score-storage (IPSS-S), interstitial cystitis symptom index (ICSI), interstitial cystitis problem index (ICPI), and visual analogue scale (VAS). We gathered these data from two private rehabilitation centers in Taiwan between January 2015 and December 2015. Ninety-five men and 11 women were enrolled in the study. The survey contained information on sex, age, age when ketamine use started, and how they used ketamine . Details of their LUTS and treatment were also recorded. Ketamine users were also asked whether they were using a combination of ketamine and other common substances, such as amphetamine, marijuana, morphine, heroin, codeine, flunitrazepam, or ecstasy. The treatments that the subjects had received, such as oral medication, hydrodistention via cystoscopy, intravesical therapy, or hyperbaric-oxygen therapy, were verified. The symptom that improved first and the most bothersome symptom were also recorded. Data processing and statistical analysis were performed using SPSS Version 17.0. (SPSS Inc. Chicago) and STATISTICA 7.


Cardinium has profound effects on the ecological adaptation of its host, the whitefly Bemisia tabaci (Gennadius) biotype Q (hereafter referred to as B. tabaci Q), the molecular mechanism underlying interactions between these two organisms is not yet fully understood. In this study, sRNA libraries were constructed, amplified, and sequenced for Cardinium-infected (C+) and uninfected (C∗−) B. tabaci Q with identical genetic backgrounds. Subsequently, the genes targeted by the differentially expressed miRNAs were predicted by integrating the B. tabaci Q genome data. A total of 125 known and 100 novel miRNAs were identified, among which 23 significant differentially expressed miRNAs were identified in both libraries. It is noteworthy that an analysis of target genes showed that Cardinium-responsive miRNA-regulated genes were functional in apoptosis, reproduction, development, immune response, thermotolerance and insecticide resistance. GO and KEGG pathway analysis revealed that some miRNA-target genes are closely associated with energy metabolism. A major finding of this study was the identification of several miRNAs that may be involved in physiological processes in response to environmental stress, i.e., insecticides and high temperatures. This information will provide a foundation to help further elucidate the functions of Cardinium in B. tabaci Q.Although the bacterial symbiont  Candidatus Cardinium hertigii (hereafter referred as Cardinium), was initially characterized in Encarsia wasps  in the exotic sweet potato whitefly, Bemisia tabaci (Gennadius) Q (hereafter referred to as B. tabaci Q) in the Shandong Province, China . We speculated that the potential negative effect of Cardinium on its host whitefly may be the result of environmental stress under field conditions, i.e., in response to insecticides and high temperatures. To date, little information is available regarding the molecular mechanism underlying the Cardinium-mediated phenotypes of their whitefly hosts  are numerous non-coding small RNAs (sRNAs) (20–24 nt) involved in the regulation of gene expression post-transcriptionally, through repression of mRNA translation or degradation of mRNA in the cytoplasm . As a re aegypti . Also, Wsymbiont . An anal females . BecauseB. tabaci B vs. Q  and an uninfected strain (abbreviated as C∗−) with identical genetic backgrounds. Deep sequencing of four sRNA libraries was then conducted for both C+ and C∗−. Subsequently, bioinformatics analyses were carried out to categorize the sRNAs, assess differential expression of known and novel miRNAs, predict their targets and perform their functional annotation. These data will provide novel insight into the mechanism of insect-symbiont interactions, especially for Cardinium in B. tabaci Q.Based on our current knowledge, sufficient data are available to characterize the profiles of miRNAs in whiteflies. Our previous results showed different expression levels of miRNAs in  B vs. Q . FurtherB. tabaci Q colony used in this study was established from individuals originally collected in the Shandong Province, China, in July 2012, and maintained in separate cultures for 3 years on potted cotton plants (Lu-Mian-Yan 21 cultivar). Whiteflies were kept in isolated screen cages under constant conditions of 27 ± 1°C, with a 16/8 h light/dark photoperiod, and 70 ± 5% RH. An introgressive backcrossing scheme was used to homogenize nuclear genetic backgrounds of infected and uninfected whiteflies for 6 generations, using the method described by + and C∗− whiteflies; the two strains were then regarded as having a nearly identical genetic background . The following morning (at 8:00 am), newly emerged adults were individually collected and transferred into plastic tubes and sexed using a stereomicroscope  with a slight modification of the procedure recommended by the manufacturer. In this procedure, RNA was purified several times with isopropanol and 75% ethyl alcohol. Total RNA (>3 μg) of good quality was used to construct an sRNA library for each sample by using a TruSeq small RNA Sample Pre Kit (Illumina). Briefly, total RNA was ligated with 5′ and 3′ adapters followed by reverse transcription using RT primers. After PCR amplification of the cDNAs, amplified PCR products within the 130–160 bp size range were purified on an 8% polyacrylamide gel . The libraries were constructed with purified RNAs and were sequenced on an Illumina HiSeq 2500/2000 platform at the Novogene Company, Beijing, China.Total RNA was isolated from the CB. tabaci Q genome1) with less than 2 bp mismatch to analyze their distribution and expression , reads with 5′ adapter contaminants, reads without 3′ adapter or the insert tags, those containing poly A, T, G, or C and low quality reads obtained from the raw data. Then, sRNA sequences of 18–35 nt were selected to conduct all downstream analyses. To prevent every unique sRNA mapping to multiple non-coding RNA (ncRNAs), we used the following priority rule: known miRNA > rRNA > tRNA > snRNA > snoRNA > repeat > gene > novel miRNA so that every unique sRNA mapped to only one annotation. The Bowtie software was used to map the sRNA tags to the pression .B. tabaci. Known miRNAs were defined as sequences that were identical to sequences from Drosophila melanogaster or other insects  as outlined previously  were estimated using the following normalization formula: Normalized expression = ,000,000 . Since t,000,000 . P-valueB. tabaci4 (We used the miRanda software to identify genes targeted by miRNAs in the genome of  tabaci4 . To furt tabaci4 .SRP076077.The dataset that was studied in this article is available in the NCBI (SRA) public repository under accession number + and C∗− whiteflies (taken from the same treatment samples as used for library construction). Reverse transcription was performed by Mir-XTM miRNA First-Strand Synthesis Kit  under the following conditions: 37°C for 1 h, 85°C for 5 min, and then held at 4°C. Levels of miRNA were assessed in four biological and two technical replicates of each sample, using SYBR Premix EX TaqTM II . The cDNA was quantified in 20μL reactions, containing 7.2 μL ddH2O, 10 μL SYBR Advantage Premix (2X), 0.8 μL miRNA-specific Primer (10 uM), mRQ 3′ Primer (supplied with the Kit No. RR820A) and 2 μL template cDNA. The primers used in this study are provided in Supplementary Table −ΔΔCt method for relative quantification of gene expression was used to determine the level of miRNA expression.Total RNA was extracted from the C+ and C∗− strains   Figure . The pro) Figure . But in nt sRNAs . This di∗− libraries (19.90%) were higher than those in the C+ libraries (13.41%), whereas the corresponding values for novel miRNAs were similar . Thus, differences of both length distribution and composition of two sRNA libraries suggest that infection by Cardinium may inhibit the synthesis of miRNAs in B. tabaci. The same results were found in T. urticae infected with the symbiont Wolbachia , novel-156 (hsa-miR-4762-3p) and tca-miR-263b-5p] were detected only in Crocesses . An earl+ strain .+ and C∗− libraries were compared after normalization . We identified 23 differentially expressed miRNAs, including 13 up-regulated miRNAs and 10 down-regulated miRNAs in the C+ strain relative to the C∗− strain  may modulate phenotypic plasticity in B. tabaci Q  annotation enrichment was performed. GO ontologies containing four different biological processes and 20 molecular functions were predicted Figure . The enrB. tabaci Q have been identified to be associated with apoptosis . The results showed that seven miRNAs had qPCR expression patterns similar to those revealed by our RNA-seq analysis in the Cin Table , while sn Figure . HoweverCardinium infection in B. tabaci Q. These miRNAs are involved in several biological processes, including cell apoptosis, reproduction and development. The major finding of this study is the identification of several miRNAs overexpressed in infected whiteflies that target genes involved in thermotolerance and insecticide resistance, pointing to a compromised resistance to heat and xenobiotic stresses in C+ individuals. These findings may help explain fitness variation among Cardinium-infected B. tabaci Q whiteflies in response to environmental stress. In general, our findings lay a solid foundation for further functional study of the interactions between Cardinium and its host whitefly, B. tabaci Q.Using high-throughput sRNA sequencing, we screened out 23 miRNAs exhibiting differential expression in response to The present research complies with all laws of the country (China) in which it was performed and was approved by the Department of Science and Technology of the Qingdao Agricultural University, China (Permit Number: 20110712).DC contributed to experimental design and management. HL carried out the data analysis and drafted the manuscript. XW and TD participated in data analysis. All authors read and approved the final manuscript.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.


WOX gene in a fern, which is located phylogenetically between the two taxa.Post-embryonic growth of land plants originates from meristems. Genetic networks in meristems maintain the stem cells and direct acquisition of cell fates. WUSCHEL-RELATED HOMEOBOX (WOX) transcription factors involved in meristem networks have only been functionally characterized in two evolutionarily distant taxa, mosses and seed plants. This report characterizes a CrWOXB transcripts were detected in proliferating tissues, including gametophyte and sporophyte meristems of Ceratopteris richardii. In addition, CrWOXB is expressed in archegonia but not the antheridia of gametophytes. Suppression of CrWOXB expression in wild-type RN3 plants by RNAi produced abnormal morphologies of gametophytes and sporophytes. The gametophytes of RNAi lines produced fewer cells, and fewer female gametes compared to wild-type. In the sporophyte generation, RNAi lines produced fewer leaves, pinnae, roots and lateral roots compared to wild-type sporophytes.CrWOXB functions to promote cell divisions and organ development in the gametophyte and sporophyte generations, respectively. CrWOXB is the first intermediate-clade WOX gene shown to function in both generations in land plants.Our results suggest that The online version of this article (10.1186/s12870-019-1991-8) contains supplementary material, which is available to authorized users. Arabidopsis thaliana, the homeobox transcription factor WUSCHEL (WUS) is a key player in shoot meristem maintenance; WUS expression is transcriptionally regulated and the protein acts non-cell-autonomously by moving from the organizing center (OC) to the central zone (CZ) of the SAM to both activate and repress gene transcription in order to maintain meristem cells in a pluripotent state [Stem cells are self-renewing pluripotent cells. In vascular plants, they are located in the shoot apical meristem (SAM) and root apical meristem (RAM). Stem cells divide at a low frequency to produce daughter cells that will either maintain the stem cell pool or actively divide and take on new identities to form new organs , 2. The nt state .WUS belongs to the family of WUSCHEL-RELATED HOMEOBOX (WOX) transcription factors, which are characterized by the presence of a conserved homeodomain [WOX genes group members into three clades: ancient, intermediate, and modern , O,, OWOX gva , P. abP. abies , 31 and a patens . Five WOichardii . RT-PCR etophyte . In situe leaves , CrWUL moot axis . CrWOXB y RT-PCR .CrWOX13A and CrWOXB in both gametophyte and sporophyte generations presents an opportunity to understand the ancestral functions of WOX proteins. In the moss P. patens, only the ancient clade of WOX genes exists, and, in contrast to A. thaliana ancient WOX genes, P. patens ancient WOX genes function in both generations [AtWOX genes, AtWOX2 and AtWOX8, are expressed in both sporophyte and gametophyte generations [AtWOX genes seem to function only in the sporophytes [Nicotiana tabacum, transcripts of two ancient and one intermediate WOX genes are found in both the gametophyte and sporophyte tissues [The unbiased expression of erations . Only twerations ; all othrophytes , 21–28.  tissues .WOX genes provide an entry point for understanding the meristem of ferns at both the development and the evolution levels. So far, the combination of WOX gene family evolution and their developmental functions has only been studied in detail in the moss P. patens and angiosperms. Similar investigation in the fern will bridge the gap in our knowledge of meristem evolution. Furthermore, comparison between the gametophyte and the sporophyte meristems within the fern will provide insight into the co-option of gene network between the meristem of the two generations. This understanding can only be fully realized with the expression and functional analyses of all five WOX fern genes. Here, we present the completed study of one of the five WOX genes found in C. richardii, CrWOXB, which is expressed in both sporophyte and gametophyte generations [CrWOXB in both generations was examined by RNAi suppression of CrWOXB expression in transgenic C. richardii plants. These results show that CrWOXB, an intermediate-clade WOX gene, is expressed in regions of cell proliferation in both the gametophyte and sporophyte. The phenotypes of the RNAi suppression lines were consistent with meristem defects, providing the first demonstration of WOX gene function in a fern.Sister clade to seed plants, ferns have sporophytic SAMs that are composed of multiple zones resembling that of the seed plants , 36. Howerations , to examCrWOXB was observed in developing gametophytes and in sporophyte leaves. CrWOXB was expressed at low but discernable levels before (d8 and d10) sexual maturation and increased in gametophytes at sexual maturity (d13) , followed by an indeterminate number of sporophylls (produce sporangia). Pinnae refer to the leaflets of both vegetative and sporophyll leaves. The expression of CrWOXB in sporophyte tissues detected by RT-PCR showed the highest expression in sporophytes with one fully expanded leaf and in the later vegetative leaves but was barely detectable in the sporophylls, the pinna-base and tip of the vegetative leaves  formed by CrWOXB RNAi suppression, crwoxb, lines, by using Agrobacterium-mediated transformation of gametophytes. Suppression of CrWOXB transcripts were quantified in young sporophytes with 6–7 fully expanded vegetative leaves by RT-qPCR. CrWOXB expression in T2 sporophyte of crwoxb lines were found to express a range of levels from a high average of ~ 40% in crwoxb10 to ~ 16% in crwoxb1 compared to wild-type plants  A. thaliana orthologs [rophytes , 43. Pri. patens . The trerthologs .CrWOXB was expressed in both the male and hermaphrodite. Expression in the male persisted briefly, during thallus growth, before cells differentiate into antheridia. Similarly, in the hermaphrodite, CrWOXB was expressed shortly after spore germination; however, its expression appeared in the notch region, during and after the emergence of the lateral meristem. The expression pattern of CrWOXB in the male and hermaphrodite is in agreement with cell proliferation regions delineated by [CrWOXB function is required soon, if not immediately, after spore germination.In the gametophyte, eated by . Our resC. richardii sporophytes, as in other ferns, can be traced back to single, apical cells [C. richardii sits atop a slender stalk of meristem cells in a region defined as the proliferation zone [CrWOXB is homogenous in the shoot, root, leaf primordia, and the vascular tissues signifying a more general role of CrWOXB in each region of cell proliferation. The homogenous expression pattern of CrWOXB in the primordia is similar to what was observed of AtWOX9 in Arabidopsis shoot apical meristem [CrWOXB in the root primordia is similar to that in the mature root tip reported by Nardmann et al. [CrWOXA, the other intermediate CrWOX gene, is expressed, suggesting some functional divergence between these two paralogs. Interestingly, CrWOXB expression was not detected in apical cell of the shoot and root but was observed in the leaf primordia  were originally obtained from Carolina Biological Supply Company . Wild-type and CrWOXB RNAi suppression lines (crwoxb) were surface sterilized in 4% sodium hypochlorite and 0.5% Tween-20 for 5 min, rinsed 4–5 times with sterile water and incubated at room temperature in the dark for 3–5 days to synchronize germination. Spores were then plated on basal media  supplemented with 100 μg ml− 1 ampicillin and maintained in humidity domes at 26 °C with a light/dark cycle of 16/8 under light intensity of 100 μM m− 2 s− 1 for gametophyte development. Plates were inverted after 10 days of growth (d10) to deter fertilization. Sporophytes were grown in BLP germination soil #1  under humidity domes in the same light and temperature regime as gametophytes.Spores of CrWOXB was cloned into vectors pK7GWIWG2 and pH7GWIWG2 to generate CrWOXB RNAi constructs using the Gateway technology as described by Curtis and Grossniklaus [Agrobacterium tumefaciens strain GV3101 from Escherichia coli with an E.coli helper strain containing the pRK 2013 plasmid [0) were selected on media containing 50 μg ml− 1 kanamycin or 5 μg ml− 1 hygromycin. Resistant gametophytes were isolated and allowed to self-fertilize to produce sporophytes (T1). Sporophytes were moved to liquid basal media and allowed to root before transplanting to soil. From the more than 20 independent transgenic lines isolated, 10 were chosen for qPCR analysis and characterization. Detailed phenotyping of three lines are presented here.A 302-bp fragment . DIC images of sectioned samples were viewed with a Nikon Eclipse E800  and captured with a Photometrics CoolSNAP cf. . To confirm gene expression patterns, each in-situ experiment was repeated at least two times using different biological samples.The SAM from young sporophytes with 10–11 vegetative leaves, the youngest a visible fiddlehead, was dissected and vacuum infiltrated with fixing solution  for 45 min and then incubated in fixing solution overnight at 4 °C. Dehydration, embedding, pre-hybridization, hybridization and post-hybridization washes were based on Jackson , except CrWOXB, and 25 cycles under the same conditions for CrUBQ transcripts.Gametophyte and sporophyte tissue were harvested and flash frozen in liquid nitrogen, then stored at − 70 °C. Total RNA was extracted from frozen tissue with the Quick-RNA MiniPrep (Plus) kit  and 750 ng of gametophyte total RNA or 500 ng of sporophyte total RNA was used in reverse transcriptase reaction using MMLV  with N9 random primers . PCR was conducted with the following cycles: 2 min at 94 °C, followed by 37 cycles of 30s at 94 °C, 30s at 59 °C, and 30s at 72 °C, with a 5 min final extension time at 72 °C for crwoxb lines, the age of the sporophytes in both the wild-type and crwoxb lines was determined by numbers of leaves and not days. Primers for qPCR are listed in Additional file CrWOXB expression was measured relative to CrUBQ.For RT-qPCR, three biological and two technical replicates were performed for each line. Total RNA from whole young sporophytes with 6–7 fully expanded round leaves was extracted and 200 ng were used in cDNA synthesis as described above. Due to delay in development of − 1)  for at least 15 min, rinsed in water and mounted on slides with 50% glycerol. Gametophytes were then imaged with a Leica stereomicroscope and a Qicam camera  with a DAPI filter. Nuclei of gametophytes were counted in Photoshop CC . Brightness and contrast were increased slightly to facilitate cell counting.To count cells of the gametophytes, gametophytes were cleared overnight in 100% ethanol at 4 °C, then rinsed 3 times for 5 min in water and stained with Hoechst 33342 .Statistical analyses of Ostreococcus tauri, Osctreococcus lucimarinus, Physcomitrella patens, Selaginella kraussiana, Oryza sativa were obtained from Phytozome [Azolla filiculoides, Salvinia cuculata sequences were obtained from Fernbase [Ceratopteris richardii sequences were obtained from NCBI. Arabidopsis thaliana sequence were obtained from TAIR. Full length protein sequences are provided in Additional file Multiple sequence alignments of WOX homeodomains are based on T-Coffee  and treehytozome . Azolla Fernbase . CeratopAdditional file 1:Figure S1. Phylogeny of WOX proteins from representative embryophytes. Abbreviations Ot, Ostreococcus tauri; Ol, Osctreococcus lucimarinus; Pp, Physcomitrella patens; Sk, Selaginella kraussiana; Af, Azolla filiculoides; Sc, Salvinia cuculata; Cr, Ceratopteris richardii; Os, Oryza sativa; At, Arabidopsis thaliana. Evolutionary history was inferred using Maximum-likelihood method with 500 bootstrap replicates as a test of relatedness. Alignment of sequences was conducted with T-Coffee and cladogram constructed in phyML and visualized with MEGA7. The WOX genes in the two sequenced fern genomes, Azolla and Salvinia [Salvinia  have notAdditional file 2:Figure S2. Bright-field microscopy of d13 transgenic gametophytes. (a) RN3 (wild type). (b-d) transgenic gametophytes. (a-d inset) 2x digital-enlargement of a ~ 250 × 250 pixel region of the notch meristem. Red lines outline the notch meristem, in (a) the prothallus overlaps and no space is present between lobes of the prothallus. Scale bar = 0.5 mm. (DOCX 12696 kb)Additional file 3:Figure S3. Protein domain comparison of intermediate clade WOX proteins from A. thaliana and C. richardii. (a) Model of intermediate clade WOX proteins from C. richardii and A. thaliana, green boxes represent the homeodomain and blue the C-terminal domain. (b) T-Coffee sequence alignment of C. richardii and A. thaliana intermediate WOX proteins domains. Specific motifs are outlined, and conserved residues are highlighted in green, less conserved sites are highlighted in yellow. (DOCX 160 kb)Additional file 4:Table S1. Primer sequences used in the study. Sequences left of * are added for directional cloning or T7 promoter sequence. All sequences are 5′ to 3′. (DOCX 20 kb)Additional file 5:Table S2. Average number of gametophyte cells observed at days 7 to 10 of growth and the average number of cells produced per day. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, Two-way ANOVA. (DOCX 20 kb)Additional file 6:Table S3. Transgenic and wild-type gametophyte cell and archegonia numbers at d13. ****, p < 0.0001. Two-way ANOVA, cell numbers or One-way ANOVA, archegonium numbers. (DOCX 20 kb)Additional file 7:Sequences used in Phylogeny. (TXT 20 kb)


To evaluate the effect of variable corneal thickness on Pentacam HR diagnostic indices in normal corneas. µm, group 2 (81 eyes) with TCT between 523 and 564 µm, while group 3 (40 eyes) enrolled TCT >564 µm. The possible effect of pachymetry on Pentacam HR indices was assessed using partial correlation tests.  Retrospective study was conducted at Al Watany Eye Hospital, Cairo, Egypt. Consecutive 160 eyes of young myopic subjects without KC were evaluated using Pentacam HR . The elevation- and thickness-based indices were recorded. Enrolled corneas were categorized into three groups according to TCT quartiles; group 1 (39 eyes) included corneas with TCT <523 P=0.013 and 0.019, respectively). Regarding pachymetric indices, maximum pachymetry progression index (PPI max) showed statistical significance (P=0.001). Partial correlations, after excluding age and refractive error effects, showed that TCT was correlated with BE from BFS, BE from BFTE, and PPI max .  In normal corneas, back elevation from best fit sphere (BE from BFS) and that from best fit toric ellipsoid (BFTE) were the elevation indices that showed statistically significant differences among groups  is recommended in relatively thin or thick corneas. In the past few decades, corneal evaluation prior to refractive surgery was performed using topography systems with “Placido-based” technology, which was considered inaccurate by some authors –3, as itPentacam HR calculates a range of indices from curvature, elevation, and pachymetric data . ThinnesRecent corneal studies aroused an important issue that deserves proper analysis. Some factors can be correlated with various Pentacam indices, and variations in these factors can significantly alter indices values and, hence, alter their interpretation. The effects of age , 17 and This is a retrospective study at Al Watany Eye Hospital, which enrolled myopic eyes of 160 consecutive subjects having normal, nonectatic corneas. All recruited participants were seeking refractive surgical correction. We excluded candidates with any detected corneal pathology, contact lens wear within the previous two weeks, narrow palpebral fissure precluding proper scanning, or previous ocular surgery. The study adhered to the tenets of the Declaration of Helsinki and was approved by the Watany Research and Development Center (WRDC) Institutional Review Board.Both eyes for each patient were evaluated using Pentacam HR, branded as Allegro Oculyzer II . Each eye was scanned according to the recommendations of the device manual (at least thrice), and the most reliable scan was chosen regarding the largest analysed area, valid data percent, and good alignment. The eye with the most reliable scan was then selected for analysis using software version 120r20.Front elevation of the thinnest point (FE) from best fit sphere (BFS)Back elevation of the thinnest point (BE) from BFSFE from best fit toric ellipsoid (BFTE)BE from BFTEElevation-based indices TCTAverage and maximum corneal pachymetry progression indices Average and maximum Ambrosio's relational thickness indices Pachymetry-based indicesThe investigated indices included the following:To keep aside the bias that could arise from the effect of age  and maniThe corneas enrolled in the study were categorized into three groups, according to TCT quartiles of this sample; each quartile comprises 25% of corneas.P value was less than 0.05.Data were collected and verified, and the compound indices were calculated. The following tests were performed using IBM SPSS Statistics : calculation of the mean, standard deviation (SD), one-way analysis of variance (ANOVA), Pearson and its nonparametric equivalent Spearman correlation coefficients, and partial correlation coefficients controlling for SE, Km, and age. Values were considered statistically significant if the µm (ranging from 463 to 648 µm).Subjects' mean (±SD) age was 27.7 ± 6.3 years (ranging from 18 to 45). SE had a mean of 4.96 ± 2.81 diopters (D) (ranging from −0.625 to −12.5), including simple myopia and/or myopic astigmatism. The mean TCT was 543.3 ± 32.4 µm (1st quartile), group 2 comprised corneas of 2nd quartile  and 3rd quartile , while group 3 (40 eyes) enrolled thick corneas of TCT between 565 and 648 µm (4th quartile).Group 1 (39 eyes) included thin corneas of TCT 463 to 522 Regarding the distribution of subjects' age, SE, and Km, they showed no statistically significant differences among groups, and consequently no bias can arise from them .The mean, SD, together with the suggested alarming values (based on mean ±2 and 3 SD) of all studied indices in the 3 groups are shown in P=0.013). The same was found regarding the BE from BFTE (P = 0.019). On the contrary, FE from BFS and BFTE did not show any statistically significant differences.BE from BFS was significantly different among groups  and than average thickness corneas “group 2” .The post hoc test showed that thick corneas “group 3” had higher statistically significant BE from BFS and BFTE than thin corneas “group 1” (P=0.001), ART avg (P < 0.001), and ART max (P < 0.001), while PPI avg showed no statistical significance (P=0.055).One-way ANOVA test detected a statistically significant difference among groups regarding PPI max (P=0.001). On the contrary, ART avg and ART max increased from group to group with the increase in thickness .The post hoc test showed that thinner corneas “group 1” had PPI max greater than thicker corneas “group 3” (P=0.001), BE from BFTE (P=0.001), and PPI max (P=0.002). On the contrary, TCT was not correlated with FE from BFS (P=0.615), FE from BFTE (P=0.626), and PPI avg (P=0.177).After exclusion of the age, refraction, and Km effects, partial correlation tests demonstrated that TCT was correlated with BE from BFS (The frequent use of Pentacam nowadays for meticulous prerefractive surgery assessment, aiming at minimizing the risk of post-LASIK ectasia, has attracted the attention of researchers in the past decade, owing to the significant negative impact of post-LASIK ectasia on visual capacity, even in the early stages of the disease . This inWe categorized our cohort of normal corneas into groups according to TCT quartiles of this sample, and also the average TCT is near the values published in the literature that evaluated TCT measurement using Pentacam –27. The These findings could be, in our view, because of two possibilities. Firstly, thick corneas may be morphologically different from thinner ones  and therSecondly, corneas with all thickness ranges may have similar morphology, but are differently assessed by the imaging device if corneal thickness values are above or below those of the adjusted normative database. This issue was previously detected in the Orbscan slit-scanning device  and may Further studies assessing the same indices using different imaging technologies, as the optical coherence tomography, may elucidate which of these two hypotheses is more plausible.We presented the 2 and 3 SD values for the evaluated indices as previously suggested . These mThis study is complementing other studies that have recently discussed a possible effect of some factors on elevation and pachymetry-based indices. For instance, the age factor has been previously discussed, evaluating its effect on topometric indices, FE and BE , and on To the best of our knowledge, this is the first study to correlate TCT with various tomographic indices. Moreover, the study suggested two possible hypotheses. Validation of our results with further studies, including larger cohorts, together with evaluating the possible existence of such correlations between pachymetry and other indices using other Scheimpflug devices rather than Pentacam HR, is highly recommended.We assume that our study has an important clinical relevance, as we highlight the possible fallacies of relying on all Pentacam indices using current normative database, without taking into consideration the pachymetric subgrouping of cohorts. These results highlight the importance of including pachymetric subgroups in Pentacam future software versions, as there is no such pachymetry subgrouping in the current software.Some Pentacam HR indices may be affected by corneal pachymetry. This necessitates the inclusion of pachymetric subgroups in Pentacam normative database. Up till then, the use of the more robust FE and PPI avg is recommended in relatively thick or thin corneas.