P < 0.001). Classic CNV presented with worse visual acuity compared with MCV at baseline (P < 0.001). Central macular subfield thickness was highest in RAP, and lowest in MCV (P = 0.036). Subfoveal choroidal thickness was highest in MCV, and lowest in RAP (P < 0.001). There was a significant difference in visual acuity at 12 months among five subtypes (P = 0.046). Our results highlight the importance of angiography for identifying AMD subtypes, particularly the novel MCV group being distinct from other subtypes.Neovascular age-related macular degeneration (AMD) is the leading cause of irreversible blindness in elderly population. Several classifications schemes have been developed to provide subtypes of neovascular AMD, which are known to be associated with visual prognosis. However, there is still a large proportion of patient with ambiguous findings according to current classification criteria. In this study, we classified treatment-naïve neovascular AMD patients using novel angiographic classification system and investigated the incidence and clinical characteristics of AMD subtypes. Among 339 eyes, five AMD subtypes were identified: 41 (12.1%) with classic choroidal neovascularization (CNV), 30 (8.8%) with occult CNV, 91 (26.8%) with microaneurysmal choroidal vasculopathy (MCV), 123 (36.3%) with polypoidal choroidal vasculopathy (PCV), and 54 (15.9%) with retinal angiomatous proliferation (RAP). MCV was younger than RAP ( There have been considerable advances in the diagnosis of AMD since Novotony and Alvis first introduced the use of fluorescein angiography (FA) in the human fundus2. Since then, several useful diagnostic systems have been introduced, including indocyanine green angiography (ICGA)3, optical coherence tomography (OCT)4, and, most recently, OCT angiography5. The advent of ICGA facilitated the detection and demarcation of occult or poorly defined choroidal neovascularization (CNV). ICGA allows retinal specialists to move beyond the present FA-based categorization of “occult/poorly defined” and “classic/well-defined” lesions to develop a more refined classification of CNV6. These new classifications include polypoidal choroidal vasculopathy (PCV)7 and retinal angiomatous proliferation (RAP)8.Age-related macular degeneration (AMD) is a progressive chronic disease of the central retina and a leading cause of vision loss worldwide9 has introduced a new standard in the treatment of neovascular AMD patients, however, the major challenge associated with anti-VEGF treatment lies in the heterogeneity of individual patient profiles. Different types of CNV are different in terms of the demographic risk profile, clinical manifestations, visual prognosis and response to treatment11. In this regard, there is a need for more consensus regarding the classification system using angiography. More specifically, there is a portion of patients that fall on the boundaries of occult CNV and PCV12. This observation may explain why the prevalence of PCV is reported differently in studies conducted on patients of the same race (22.3–61.6%)13. Therefore, it is important to redefine the classification criteria using angiography, ultimately in order to refine present and future treatment strategies to improve visual outcomes.The advent of intravitreal therapy using anti-vascular endothelial growth factor (VEGF)Therefore, the aim of the present study was to determine the incidence and clinical characteristics of angiographic subtypes of neovascular AMD including microaneurysmal choroidal vasculopathy – a novel subtype of AMD that we hypothesized as a distinctive entity from PCV and occult CNV - and compare functional outcomes with other subtypes.We identified 353 eyes from 304 patients with neovascular AMD based on their electronic records. Fourteen eyes were excluded because they had vitreous hemorrhages (n = 6), or because of an absence of ICGA photographs at baseline (n = 8). Ultimately, 339 eyes from 290 patients with neovascular AMD were included in this study. The mean age of the patients was 72.4 ± 8.0 years , and 59.0% were male Table . The mosBased on the FA findings, 105 eyes (31.0%) were suspected of having classic CNV and 234 eyes (69.0%) occult CNV. Those classified with occult CNV includes patients with occult with minimally classic CNV. Of the eyes with “classic CNV on FA,” 42 eyes (40.0%) were diagnosed with PCV, 41 (39.0%) with classic CNV, 15 (14.3%) with MCV, and 7 (6.7%) with RAP on ICGA. Of the eyes with “occult CNV on FA,” 81 eyes (34.6%) were diagnosed with PCV, 76 (32.5%) with MCV, 30 (12.8%) with occult CNV, and 47 (20.1%) with RAP on ICGA.Eyes in the MCV, PCV, and RAP groups had a higher proportion of hot spots than did those in the other groups . Polyps were identified after there was pneumatic displacement of submacular hemorrhages in 5 eyes in the PCV group. In these cases, the polyps were not visible at baseline. BVN were detected in 14.6% of classic CNV, 6.7% of occult CNV, 100% of MCV, 84.6% of PCV, and 5.6% of RAP patients (P < 0.001). Plaques were detected in the late phase of ICGA in 40.0% of occult CNV and 20.4% of RAP cases, which differed significantly from those in the other groups (P < 0.001).μm, 328.9 ± 108.0 μm, 298.4 ± 87.7 μm, 309.5 ± 87.5 μm, and 348.9 ± 133.8 μm, respectively (P = 0.036). The subfoveal choroidal thickness in classic CNV, occult CNV, MCV, PCV, and RAP were 145.5 ± 85.1 μm, 183.5 ± 61.9 μm, 273.8 ± 105.3 μm, 232.8 ± 88.8 μm, and 125.4 ± 51.4 μm, respectively   and MCV (26.8%). Classic CNV was identified in 12.1% of eyes, occult CNV in 8.8%, and RAP in 15.9%. The sum of eyes with MCV and PCV exceeded 60% of all eyes with neovascular AMD. However, the proportion of RAP was similar to that of previous studies17. According to the criteria of the EVEREST study18, MCV should belong to the category of PCV. However, it could have been classified as a part of occult CNV or chronic CSC in the preceding studies19, as there is no definite ‘visible’ polyp observed on ICGA. This finding may explain the discrepancy in the proportion of PCV across studies, even those focusing on the same races20. In this regard, this novel classification system may contribute to improved categorization of neovascular AMD patients. This method is strengthened by its comparatively high ratio of inter-observer correlation.This study provides several important issues in investigating the neovascular AMD. We can estimate the proportions of each subtype of neovascular AMD among the Asian population. When classified using FA only, the proportions of classic CNV and occult CNV were 31.0% and 69.0%, respectively. This incidence is similar to previous study, in which classic CNV was found in 23% of eyes and occult CNV in 71%et al. reported that the prevalence of RAP is between 10% and 15%, with a female and Caucasian predominance16. The frequency of RAP in this study (15.9%) is consistent with findings reported in similar studies on Caucasians, but is higher than that reported in Asian populations (4.5%)22. Lee et al.23 also reported that the rate of RAP was high in Koreans (25%). Interracial differences in the proportion of neovascular AMD can be expected, because the Y402H variant plays a major role in the etiology of AMD and is obviously different between Caucasians and Asians24. Most Korean patients do not have the Y402H variant. Therefore, there are likely other, yet unidentified, genetic variants that also promote disease progression in RAP.Another advantage of this study is that we can compare the proportion of subtypes classified using FA and ICGA, respectively. Of the classic CNV patients diagnosed using FA, only 39.0% were proper classic CNV, compared to just 12.8% in the occult CNV. While FA was useful to assess leakage from CNV lesions, it was insufficient for demarcating occult or poorly defined CNV, which is necessary for identifying PCV and MCV. Yannuzzi 19. Moreover, Uzawa et al.25 and Tan et al.26 advocated that the PCV is not a homogenous disease entity, but a spectrum of disease with discriminative characteristics. Previous study also revealed a lower anti-VEGF treatment response associated with thicker choroids in PCV patients27. These results are likely to be caused by the coexistence of different subtypes within the category we classify as PCV. In this regard, the refined classification system including MCV may reduce such debate and clarify the subtype of neovascular AMD. MCV is distinct from PCV by the absence of definite polyps. It is distinct from occult CNV by the presence of microaneurysms, which are connected with a lacy choroidal vasculature on ICGA , diabetic retinopathy, retinal vasculopathy, inflammatory diseases, intraocular tumors, history of intraocular surgery (other than cataract surgery), and other ocular diseases that could influence visual function.Our baseline investigations were as follows: an examination for the Snellen best-corrected visual acuity (BCVA) and anterior segment by slit lamp; dilated fundus examination; and confocal scanning laser ophthalmoscopy (SLO) FA and ICGA using eye-tracked Spectralis Heidelberg Retina Angiograph (HRA) + OCT . Angiograms were obtained using a standardized imaging protocol. The early phases (up to 60 s) were imaged using dynamic ICGA and FA. Consecutive photographs were then taken for up to 1 min, and again at approximately 2.5, 5, 10, 15 and 20 minutes. These images were reviewed using Heidelberg Eye Explorer software (V.1.7.0.0). We also obtained simultaneous SD-OCT cross-sectional images, which correspond to either the site of the hot spot, plaque, or the network vessels on SLO. In addition, horizontal and vertical cross-sectional enhanced depth imaging (EDI)-OCT was performed at the fovea.Classic CNV was defined by discrete, well-demarcated focal areas of hyperfluorescence that can be discerned in the early phases of the FA. Hyperflourescence increases in intensity and extends beyond the boundaries of the hyperfluorescent area, which is identified in earlier phases of the angiogram of mid- and late-phase frames.Occult CNV was defined by late choroidal-based leakage in the early or mid-phase FA to account for an area of leakage in the late phase. There was no clearly identifiable classic CNV in FA or the presence of a polypoidal lesion, vascular network, or anastomosis in ICGA.Microaneurysmal choroidal vasculopathy (MCV) was defined by the presence of early multiple focal hyperfluorescent areas (appearing within the first 6 minutes after injection of indocyanine green) in ICGA. These areas consist of microaneurysms, which are smaller than the secondary branch of major retinal vein width, and a branching vascular network (BVN) that fills simultaneously during the choroidal filling phase.Polypoidal choroidal vasculopathy (PCV) was defined by the presence of early subretinal focal ICGA hyperfluorescence with a definite polyp. In addition, there was at least one of the following angiographic or clinical criteria: (i) association with a BVN; (ii) nodular appearance when viewed stereoscopically; (iii) presence of a hypofluorescent halo (in first 6 minutes); (iv) orange subretinal nodules on a stereoscopic color fundus photograph (polyp corresponding to ICGA lesions); or (v) association with massive submacular hemorrhage (defined by a hemorrhage of at least four disk areas).Retinal angiomatous proliferation (RAP) was defined by the subretinal, intraretinal, or preretinal hemorrhages or by retinal edema, dilated retinal vessels, retinal–retinal anastomosis, sudden termination of the retinal vessels, a ring of lipid exudates and PED, and retinal–choroidal anastomosis in dynamic FA and ICGA.We devised a classification system of neovascular AMD based on prior observation of patients from our clinic. Two independent retinal specialists (K.B. & E.S.K.) who were blinded to the treatments classified the angiographic findings into one of five types Fig. . In case29. We defined patients in whom subretinal or intraretinal fluid was present or increased, even in the presence of monthly anti-VEGF injections, as non-response. Active polyps, as well as lesions that were non-responsive to 3 consecutive anti-VEGF injections, were treated using photodynamic therapy (PDT) with verteporfin  guided by ICGA. Full-dose, full-duration PDT was performed. The treatment zone only included the visible polyps in PCV and active CNV lesions in other categories. The treatments were initiated or repeated when there were clinical signs of disease activity, such as a drop in BCVA of ≥1 line, subretinal fluid or macular edema, or evidence of active CNV on angiography.Patients were treated using anti-VEGF agents  by two retinal specialists (E.S.K. and S.Y.Y.) using a standardized treat-and-extent protocolWe recorded the patient age, sex, BCVA, and the lesion type at the time of the first injection by ICGA. During the follow-up visits at 3, 6, 9, and 12 months after the initial treatment, all patients underwent BCVA measurement, dilated fundus examination, and SD-OCT. At each visit, all of the treatments were recorded, along with the BCVA and central subfield thickness of the macula.The following parameters were used to evaluate the SD-OCT images: the presence and type of RPE detachment, subretinal fibrosis, atrophy of the ellipsoid zone, and subfoveal choroidal thickness.The primary outcome was the difference in the baseline characteristics of the subtypes of neovascular AMD based on novel classification. The secondary outcomes included the mean BCVA and central subfield thickness 12 months after initiating treatment, and the number of intravitreal injections.Statistical analyses were performed using SPSS version 24 . Interobserver agreements were evaluated using the intraclass correlation coefficient (ICC) value. The Snellen BCVA was converted into logarithm of the minimal angle of resolution (LogMAR) units prior to the analysis. The chi-square test was used to compare categorical variables, while the one-way analysis of variance (ANOVA) was used for continuous variables. Appropriate parametric analyses were performed when the data had a normal distribution. P-values less than 0.05 were considered statistically significant.Supplementary table 1


Takotsubo cardiomyopathy is characterized by chest symptoms, electrocardiographic changes, and new regional wall motion abnormality in the apical segment of the left ventricle in the absence of obstructive coronary artery disease. Particularly, apical ballooning is broadly recognized as the classic form of takotsubo cardiomyopathy. Although the prognosis of most patients with takotsubo cardiomyopathy is generally favorable, complications associated with the morphological features of transient apical ballooning are not uncommon.We describe two cases of transient complications in postmenopausal patients with takotsubo cardiomyopathy. Intraventricular thrombus was observed in Asian patient 1, and severe mitral regurgitation was observed in Asian patient 2. These complications were confirmed by transthoracic echocardiography immediately after typical takotsubo cardiomyopathy with apical ballooning was diagnosed. Anticoagulant therapy with heparin and warfarin was continued for 1 week in patient 1. After the therapy, complete resolution of the apical thrombus and recovery of systolic function of the left ventricle was observed by follow-up transthoracic echocardiography. In patient 2, transthoracic echocardiography indicated significant mitral regurgitation, which was caused by left ventricular tethering of the anterior mitral leaflet rather than left ventricular outflow tract obstruction or systolic anterior motion. Because the hemodynamic stability in patient 2 had been preserved, she was managed with conservative treatment. After approximately 1 month, follow-up transthoracic echocardiography revealed that mitral regurgitation had almost disappeared with complete resolution of left ventricular wall motion abnormalities.The presented cases indicated that important complications, such as intraventricular thrombus and severe mitral regurgitation, are associated with takotsubo cardiomyopathy in the acute phase. Because these complications are risk factors for developing a thromboembolic event or heart failure and/or pulmonary edema, timely and accurate identification of these complications is critical to achieving optimal clinical outcomes in patients with takotsubo cardiomyopathy. Takotsubo cardiomyopathy (TCM), also known as stress cardiomyopathy or broken heart syndrome, is typically characterized by the morphological features of transient apical ballooning of the left ventricle. It occurs more often in postmenopausal women and is frequently associated with emotional, psychological, or physical stress preceding the presentation . CommonlRecently, TCM has been recognized worldwide, and reports on patients with TCM have been increasing, but there are only a few reports on thrombosis and valvular disease complicated with TCM. We report two cases of patients with TCM with transient complications, such as formation of an intraventricular thrombus and severe mitral valve regurgitation (MR).2). Her blood pressure was 178/86 mmHg, pulse 99 beats/minute (regular), and respiratory rate of 18/minute with oxygen saturation of 96% on room air. Her body temperature was 37.2 °C. Her physiological examination revealed no abnormalities, with the exception of arm pain. She had alert consciousness, and her neurological examination result was normal. On the day following admission, she experienced sudden onset of chest pain and palpitations associated with cold sweats and shivering. On physical examination, her heart rate, blood pressure level, and oxygen saturation were 118 beats/minute, 119/75 mmHg, and 97%, respectively. She was afebrile, and neither heart murmurs nor abnormal breath sounds were heard. Her abdominal examination showed no notable findings. Edema was not detected in either lower limb. Her consciousness level was clear, and no apparent neurological deficit was observed. An electrocardiogram (ECG) showed ST-segment elevations in II, III, aVF, and V3–V6 leads. Her creatine kinase (CK) and N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels were 519 U/L  and 5435 pg/ml (< 125 pg/ml), respectively. Her qualitative troponin T was positive. Two-dimensional TTE using speckle tracking  with an automated function imaging technique provided with the commercial imaging analysis software  showed akinesis of the middle and apical segments of the left ventricle with typical apical ballooning and hyperkinesis of the basal segments  and lansoprazole 15 mg). She had no smoking habit or alcohol consumption. She also had no family history or employment history of note. On examination at the time of admission, her height and weight were 1.45 m and 43.0 kg, respectively , 119/84 mmHg, and 100%, respectively. Her body temperature was 36.2 °C. A grade 3/6 holosystolic murmur could be heard at the apical heart area as the loudest, whereas friction rubs and gallops were not heard. The rest of the examinations, including respiration and abdomen, were unremarkable. Edema was not detected in either lower limb. She was awake, alert, and oriented. Her neurological examination on admission did not reveal any motor or sensory deficit, and her cranial nerves were normal. ECG showed ST-segment elevation in V5 and V6 leads. The main laboratory findings were as follows: troponin T 1.04 μg/ml , CK 543 mg/dl (43–165 mg/dl), and NT-proBNP 2441 pg/ml (< 125 pg/ml). TTE showed apical LV wall akinesis with basal hyperkinesis and severe MR. She underwent an emergent cardiac catheterization. Coronary angiography showed no obstruction, and left ventriculography revealed an ejection fraction (EF) of 67.8%, severe localized apical hypokinesis with hyperkinesis of the basal segment, and grade 3 severe MR. Further two-dimensional echocardiographic examination with speckle tracking on the admission day demonstrated severe LV apical systolic dysfunction   or myocardial infarction (4.6%) [et al. reported a case of a patient with TCM-related LV protruding thrombus requiring surgery [The incidence of cardiac thrombus due to TCM is reported to be 2.2–8.0% , 6. In mgulation . Furthergulation . These mn (4.6%) . A reporn (4.6%) . The thrn (4.6%) . A muraln (4.6%) . Althoug surgery .Significant (moderate-to-severe or severe) acute MR is another potentially serious complication, accounting for 8–19% of patients with TCM , 17. In There are no randomized clinical trials to establish specific treatment of TCM. In patients with mild TCM, no treatment or a short course of limited medical therapy may be sufficient. Renin-angiotensin-aldosterone system blockers may be considered during the period when regional wall motion abnormality is present in patients with heart failure with reduced EF. Administration of β-blockers would be reasonable when coronary spasm is not suspected as the etiology of TCM, because catecholamine is often excessive. When the patients with TCM have severe congestive heart failure or cardiogenic shock, it is important to assess whether significant MR or LVOT obstruction is associated with their condition. Cardiogenic shock, which occurs in 4–20% of patients with TCM, may be exacerbated by right ventricular involvement, LVOT obstruction, or acute MR . In patiIntracardiac thrombus and severe MR may be causes of embolic events and cardiogenic shock, respectively, which may lead to poor clinical outcomes. Therefore, detection and diagnosis of the presence of complications, especially in the acute phase, and appropriate treatment of the complications are necessary for patients with TCM.


Ocimum basilicum L.), peppermint (Mentha × piperita L.) and sage  was also presented. The half-lives of CLE and SPI in herbs were 1.10–1.56 days and 0.51–0.83 days, respectively. The terminal residues of SPI  and CLE (CLE-sulfone and CLE-sulfoxide) in herbal matrices were measured below EU maximum residue limits. In this paper, we aimed to assess the impact of washing and dehydratation pretreatment and calculated processing factors (PFs) which can be applied to more accurate food safety assessments. The PF values of CLE and SPI after drying prior washing was below 1 indicating reduction of initial residues. Drying process without washing demonstrated increases of SPI concentrations (PF up to 1.50). The lowest PFs were obtained when raw herbal plants were washed before drying showing almost complete degradation of parent compound (93–99%).Herbs may contain pesticide residues which are an important discriminator of food security and food quality. The challenge of the research was to assess the fate of the herbicide clethodim (CLE) and the insecticide spirotetramat (SPI) applied in herbs (BBCH 11-21) during herb growth and processing under controlled greenhouse trial conditions. The metabolic profile of CLE and SPI and their degradation products in basil ( These include phenolic compounds, alkaloids, flavonoids, anthocyanidins, cardenolides or tannins2. In addition to these essential and valuable substances, herbs contain contaminants, especially pesticide residues which are a critical discriminator of food quality and safety3. Pesticides belong to substances toxic for humans and some of them are mutagenic, teratogenic, carcinogenic and allergenic and persistent in the environment 4.Herbal plants are widely used fresh materials for the food and health industries, especially dried herbs and spices are popular for consumers because they have multiple phytochemical properties8. According to a report from the European Food Safety Authority9, 65.1% of the analyzed herbs contained mainly organophosphate and pyrethroid insecticides, 9.4% exceeded the Maximum Residue Limits set by the EU . In herbal samples, insecticides , fungicides and herbicides are detected at average concentration levels from 0.01 mg/kg up to 2.0 mg/kg15.The maximum acceptable levels have been unified by various international organizations to assure the safety of raw herb commodities used as pharmaceutical products or food16. In some instances metabolites are more toxic and consequently pose a greater risk to the environment than the parent compound. Clethodim and spirotetramat both have a complex residue definition  SPI-enol, SPI-ketohydroxy, SPI-monohydroxy and SPI-enol-glucoside). Only limited information on the toxicological properties of CLE/SPI-metabolites is available and are presented in citrus, Appiacea and Brassicaceae vegetables, colza and soil18. Thus, it is of great significance to evaluate the metabolic profile of CLE and SPI and their behavior during plant growth and processing. The combined exposure of CLE/ SPI and contribution of individual metabolites is needed in the toxicological characterization to make more accurate food safety assessments.Clethodim is cyclohexanedione herbicide very often used for the selective control of the monocotyledonous weeds such as millet, thick oats in herbal plants. Insecticide, spirotetramat belongs to tetramic acid family and is very commonly used against aphids because herbal plants are very often attracted by this insect Table . After t19. The World Health Organization (WHO) survey presented that most populations rely on non-conventional herbal medicines. Despite the presence of many health benefits of herbs, many contaminates in fresh commodities during cultivation, harvesting, processing and storage can occur and make their consumption unsafe19.Herbs are commonly used medicinal means as a complementary or an alternative form of healthcare and are considered to be safe22. It might completely reduce the concentration, have no lack of influence and sometimes increase the levels in the final product25. Herbal raw materials, due to their short durability and seasonality, undergo various thermal treatments. Drying is the oldest method of preservation of food and the most frequently used way of preserving herbs that is the basis for their further use. It allows extending the durability of herbal products, preserving their taste, aroma, as well as many pro-health properties27.Technological processing is the method that changes the concentration of toxic substances in the processed product28. Drying significantly reduces pesticide residues, regardless of whether the material is exposed to solar radiation or using drying equipment29. Thus, development of processing technologies for reduction of toxic substances in herbal commodities was a priority for this research.The improvement of living standards showed that people increasingly care more about their health and pay attention to food safety. Herbs, especially in the dried form of e.g. spices, teas, pharmaceutical and cosmetics product are very popular for consumers31. The above facts clearly indicate that nowadays, it is an extremely important goal to evaluate the behavior of clethodim and spirotetramat.To our best knowledge, the effect of food processing on CLE/SPI in herbs was not studied. The scientific literature is focused on individual groups of compounds (mainly organophosphorus) during the preparation of herbal infusions or cooking herbsOcimum basilicum L., Mentha × piperita L. and Salvia officinalis L.) and determine the half-lives; (3) investigate the residual of these pesticides and metabolites during processing of fresh herbal plants; (4) calculate the PFs of sum CLE and sum SPI in washing/drying process regarding to EU residue definition and (5) an additional scientific aspect of the work will be supplementation PFs databases for the first time for herbs.Therefore, the science aim of the study was to: (1) evaluate metabolic profile of the cyclohexanedione herbicide and the tetramic acid insecticide in herbs during plant growth; (2) present dissipation dynamics of CLE and SPI in fresh basil, peppermint and sage  was 0.005 mg/kg whereas limit of quantification (LOQ) was 0.001 mg/kg for spirotetramat and clethodim and their metabolites.A modified QuEChERS method for simultaneous determination of SPI and CLE and their six metabolites in various species of herbal plants was applied followed by LC-MS/MS. The average recoveries of spirotetramat/SPI-metabolites in fresh and dried matrices were within 85–101% and 87–96%, respectively while for clethodim/CLE-metabolites in the range of 80–108% and 74–102%, respectively. These values were within the range expected for residue analysis. The reproducibility of recovery results, as indicated by relative standard deviations (RSDs) < 20%, confirmed that the method is sufficiently reliable for pesticide analysis in this studyThe initial residues of herbicide clethodim after 1 h of application in basil, peppermint and sage were 3.897 mg/kg, 4.251 mg/kg and 4.197 mg/kg, respectively  with dissipation equation: y = 2,6677e−0,443x in basil; y = 2,5637e−0,688x in peppermint and y = 4,5925e−0,527x in sage and the half-life of clethodim was 1.56, 1.10 and 1.38 days. Dissipation pathway of CLE in soil samples collected from the herbal plants cultivation proved that CLE rapidly converted to its sulfoxide in all cases (CLE and CLE-sulfone residues lower than LOQ). The initial concentration of CLE-sulfoxide in soil was below 0.250 mg/kg and reduced to below 0.010 mg/kg (more than 95%) after 21 days after application with the half-lives in the range of 4.1–4.5 days.Fate of clethodim and its metabolites in three species of herbal plants is presented in Fig. et al.17 who showed that clethodim sulfoxide dissipated quickly in rape plant and soil with half-lives of about 4–4.3 days. CLE-sulphone similarly increased initially then decreased in rape plant but was not detected in soil. According to PPDB33 clethodim degradation is 3 days in soil (DT50 field). Clethodim sulfoxide and sulphone are mainly formed in soil with estimated maximum occurrence fraction 0.76 and 0.16, respectively, Interestingly, Sandin-Espana et al.34 reported very rapid photodegradation of clethodim and and its metabolite sethoxydim obtained on silica gel plates in soil and plant surface model systems under simulated solar radiation with half-lives of 1.8 min and 5.0 min, respectively.Our results are consistent with You The initial residues of insecticide spirotetramat in basil, peppermint and sage after 1 h of application were 2.619 mg/kg, 3.830 mg/kg and 3.904 mg/kg, respectively  in herbs were lower than EU MRL 4.00 mg/kg. Finally, the concentration of basil, peppermint and sage decreased to 0.196 mg/kg, 0.030 and 0.125 mg/kg, respectively, which indicated that up to 99% of the initial concentrations reduced over the 21 days. Residues dissipated very fast during initial period followed by the first-order equation which provided the best fit to experimental data: y = 35.734eAs presented in the Fig. et al.35 showed the first order model for the dissipation of spirotetramat. The half-lives in citrus and soil were in the range of 2.3–8.5 days. However, Łozowicka et al.18 reported DT50 values of the SPI in Appiacea and Brassicaceae vegetable roots and leaves were 2.8–2.9 days and 2.1–2.4 days and in soil 0.2 day, respectively.Similar results reported Zhang The change of concentration levels of SPI and CLE were investigated during three processes: washing, drying and washing with drying. The processing factors were assessed for each parent compound, metabolite and also taking into account a sum of parent compound and metabolites expressed as a total residue Table .Table 3P27.The main purpose of the drying operation is to efficiently decrease the high water content of the harvested spices to a safe level in order to get a stable, safe and good quality productThe processing factor (PF) is the main parameter indicating the changes of pesticide residue level during processing. The calculated PF values for total clethodim after processing were all less than 1 (PFs = 0.14–0.54) indicating reduction in the processed herbs. The initial concentrations of CLE and its two metabolites indicated that 5–6% of clethodim transformed to clethodim sulfone and 46–53% to clethodim sulfoxide. The processing factors for spirotetramat total were calculated for parent compound and its two metabolites B-ketohydroxy and B-enol because B- enol glucoside and B-monohydroxy were not detected in processed samples. The initial concentrations of spirotetramat and its metabolites indicated that 2–4% of spirotetramat transformed to its B-ketohydroxy and 48–58% to B-enol metabolite. PF values of total spirotetramat were in the range of PFs = 0.27–1.50.36. The effectiveness of washing depends on pesticide solubility in water or in different chemical solvents. Thus, clethodim has a good penetrating ability due to its hydrophilic property characterized by high solubility in water (5450 mg/L) and was more easily to remove from basil, peppermint and sage with reduction up to 51% (PFs = 0.49–0.54) than slighty soluble spirotetramat  with the loss of residues from 32% to 41% (PFs = 0.59–0.68). The obtained results are consistent with our previous work, in which we have demonstrated that the solubility played a significant role in pesticide removal. Compounds soluble in water (e.g. acetamiprid 2950 mg/L PF = 0.43) were more susceptible to elimination during washing fruit and vegetables in contrast to insoluble lambda-cyhalothrin 37.Herbs, like other food, undergo the washing step prior to consumption or further processing, it could be expected for pesticides, especially those with limited movement and penetration ability and with a high tendency of loosely maintaining at outer surface, to be removed with reasonable efficiency by this preparatory step29. This procedure demonstrated different effectiveness because it has been found to reduce clethodim and clethodim sulfoxide residues and concentrate clethodim sulfone (PFs > 2). However, total clethodim were in the range of PFs = 0.36–0.38. Meanwhile, spirotetramat and spirotetramat B-ketohydroxy residues were concentrated . The recorded decreases in pesticides were attributed to evaporation of their residues during drying, while the increase in residue levels was most likely due to weight changes during the process.Drying is a simple traditional method of herbs preservationet al.38 who reported that pesticide concentration in processed agricultural product is higher than in raw agricultural product because drying leads to vaporization of water. In our earlier experiment, we have also reported processing factors above one for three pyrethroids (up to PF = 1.76) subjecting berry fruit the high temperatures37. Thermal processing did not affect the reduction of alpha-cypermethrin, deltamethrin and lambda-cyhalothrin, compounds with very high degradation point (from 248 °C to 275 °C).This phenomenon may be explained due to temperature in hot air drying caused thermal decomposition of clethodim . However, spirotetramat PF values of drying were more than 1. Spirotetramat  residue content in dried herbs tended to increase because of volatilization of water during processing. This is with agreement with research of Kim Interestingly, the lowest PFs values in this study were observed when raw commodity was washed before drying. The greatest loss of clethodim and spirotetramat was achieved after drying proceeded by washing, up to 99% . The residues of clethodim sulfoxide and SPI-enol residues were reduced up to 81% and 70%, respectively. However, clethodim sulfone and SPI-ketoxydroxy after drying regardless of the use of washing in preparation of herbs were more than those in raw plants showing concentration factor above 1. The overall results showed that total clethodim and total spirotetramat residues in dried basil, peppermint and sage were lower than those in unprocessed herbs .22 who reported that the concentration of pesticide in processed product is related to solubility, volatility, polarity and thermal decomposition. The loss of residue would mainly attribute to factors of degradation, evaporation and co-distillation39. Thus, Lee40 reported that hot air drying eliminated 20–30% of azoxystrobin in ginseng products, while Mergnat et al.41 found that industrial dehydration reduced phosalone levels in apples by over 80%.The results demonstrated that washing played very important role in removing residues. This explains that water soluble clethodim  was more easily removed than spirotetramat . The larger scope of processing factors proved washing prior to starting the drying process was effective step in removing pesticides. Many factors affected the removal of residue levels e.g. physicochemical properties and systemic character of the pesticides and allowed to make assumptions to explain the difference in the processing factors. These results were similar to the findings of Keikotlhaile37. Special attention has been paid to finding the relationship between the efficiency of the technological process and selected physico-chemical properties of active substances. We proved that the key parameters affecting the removal effectiveness were physico-chemical properties of substance i.e. solubility, polarity, degradation point.Previously, we created a comprehensive database on 160 PFs (processing/pesticide/product combination) for fruit and vegetables after different processing treatments previously. Water (PFs = 0.09–0.94), mechanical (PFs = 0.13–0.32) and thermal (PFs = 0.02–0.57) processing were very effective in reduction of pesticides in berry fruits, Brassica and fruit vegetables42. Thus, combination of washing prior drying is very important practice in herbal plant preservation.The results of the present research focused on herbal matrices might complement European databases of PFs  which contain data mainly for fruit/vegetables. The overall PFs for CLE-total and SPI-total in the final herb product were in the range of PFs = 0.14–0.18 and PFs = 0.27–0.31, respectively. Significant reduction of the initial concentration levels in basil, peppermint and sage in the range of 69–86% indicates that these herbs are safer to consumersIn the present work, valuable information regarding the metabolic profile of herbicide CLE and insecticide SPI in herbs during plants growing and processing was provided. Our findings predict the safe application of clethodim and spirotetramat at the recommended dosage to protect herbal plants and provide more understanding of herbicide and insecticide behavior in herbs.These results will supplement the PF database in reference to herbs and might provide more accurate risk assessments of the cyclohexanedione herbicide and the tetramic acid insecticide.The analytical standards of CLE and its two metabolites CLE sulfone, CLE sulfoxide, and SPI and its four metabolites SPI-enol, SPI-keto-hydroxy, SPI-mono-hydroxy, SPI-enol-glucoside (<99.0% purity) were obtained from Dr. Ehrenstorfer . All reagents used pesticide residue grade and were obtained from J.T. Baker . More details in Supplementary Information.2 experimental plots, which were separated by a 1 m buffer zone between plots to minimize possible cross-contamination between treatments. The treatment included herbal dynamic test treatment (fate), three terminal residual test treatments (processing) and one control (without pesticides) ) and spirotetramat (100 kg/m3 (9.35%)) were applied on basil (Ocimum basilicum L.), peppermint (Mentha × piperita L.) and sage  were sprayed with manual sprayer at the recommended dose  during herb grow at leaf development stage (BBCH 11-29 from the first leaf stage to the end of the tillering phase). The temperature in the greenhouse ranged from 14 to 33 °C and humidity ranged from 75% to 100% from the day of spraying until harvest. The samples from each treatment were collected separately  Fig. . ClethodThe herbal plants immediately after collecting were brought to the analytical laboratory in the same day. The samples for dissipation kinetics without any processing operation were thoroughly homogenized using laboratory blender. The samples for processing study were processed within 24 h after being collected from the greenhouse to determine the changes in clethodim and spirotetramt concentrations in herbs , drying (D), washing and drying (W + D). For this purpose, samples immediately after sampling were subjected to washing prior drying process, with and without previous pre-treatment Fig. . Herbs w43. Analytical procedure was previously reported by Rutkowska et al.44. More details in Supplementary Information.The modified quick, easy, cheap, effective, rugged and safe (QuEChERS) method was used for extraction and purification of pesticide residues in fresh/processed herbal and soil samples according to EN 15662:201818 column  (Phenomenex) analytical column, maintained at 40 °C during the experiments system was operated. More details in Supplementary Information.Chromatographic separation was carried out with an Eksigent Ultra LC-100  liquid chromatography (LC-MS/MS) equipped with a Kinetex XB-C2) at five points ranging from 0.005 to 2.0 mg/kg. The sensitivity was evaluated by determining the limit of detection (LOD) and the limit of quantification (LOQ) according to Document No. SANTE/1813/201734. More details in Supplementary Information.Recovery test was performed using spiked herb samples (fresh/dried) at four different concentration levels of selected pesticides  in triplicate. Linearity was assessed via the determination coefficient (R1/2) was measured. The following two equations were used: Eq. 1. Ct = C0e−kt and Eq. 2. t1/2 = ln2/k, where Ct - concentration of the pesticide residues (mg/kg) at time t  and k - the dissipation rate constant (per day).The dissipation kinetics of CLE and SPI during different species herbal plants growth was estimated using first-order kinetics equation. The persistence of each compound expressed in terms of half-life  as follows: Processing factor (PF = Residue after processing (mg/kg)/Residue before processing (mg/kg). The PF values below 1  indicate a reduction of residues in a processed commodity, whereas the values above 1  indicate concentration effects from the processing procedures. The unprocessed herb samples obtained from supervised field trial with initial deposits of pesticides were necessary to calculate the processing factors.Supplementary Information.


Pancreas specimens were analyzed after sacrifice for adipocyte infiltration, fibrosis, acinar-ductal metaplasia, abnormality of Langerhans islets , and hemosiderin deposits in acinar or endocrine locations. We found that HFD6 rats had more fibro-inflammatory islets (P = 0.0139) and acinar-ducal metaplasia (P = 0.0843) than HFD3 rats. Rats with HFD3+6 had more fibro-inflammatory islets (P < 0.0001), hemosiderin deposits (p < 0.0001), fat infiltration (P = 0.0008) and acinar-ductal metaplasia lesions (P = 0.0424). Weight increase was associated with glycoregulation abnormalities  and adipocyte infiltrations (P = 0.009). After surgery, less fibro-inflammatory islets (P = 0.0004), fat and iron infiltrates (P = 0.005 and P = 0.06), and acino-ductal metaplasia (P = 0.05) were observed compared to HFD6 rats. MR image quantifications revealed increased elasticity, fat fraction, and R2 and a decreased elasticity wave dispersion coefficient in the high fat groups that reversed after surgery. MRI parameters were in strong correlation with respective histological counterparts. In conclusion, obese rats develop pancreatic inflammatory lesions with acinar-ductal metaplasia in acinar location and the endocrine-exocrine interface. These changes can be prevented by bariatric surgery. Quantitative MR imaging is accurate in identifying early pancreatic lesions.Obesity is a risk factor for pancreatic diseases. Bariatric surgery is one of the most efficient treatments of morbid obesity. The aims were to assess pancreatic endocrine and exocrine lesions in obese rats, to analyze effects of bariatric surgery. Sixty-three male Wistar rats were included in five groups: 2 fed with high fat diet (HFD) or normal diet for 3 months, 2 fed with HFD or normal diet for 6 months; 1 group fed with HFD and undergoing bariatric surgery (n = 30). Quantitative MR imaging was performed in HFD The role of obesity in the spectrum of pancreatic diseases is still controversial. In 2014, a meta-analysis of factors predisposing to pancreatic diseases (acute and chronic pancreatitis and pancreatic cancer) in the general population included 51 population-based studies with more than 3 million individuals and nearly 11,000 patients with pancreatic diseases. Tobacco use was the most important risk factor for pancreatic diseases (relative risk (RR), 1.87; 95% confidence interval (CI), 1.54–2.27), followed by obesity  and heavy use of alcohol 2. Numerous epidemiological studies confirmed that obesity is a risk factor of pancreatic cancer in obese men and women (Body mass index (BMI), kg/m2 ≥30.0), with a relative risk estimated to 1.76  and 1.70 , respectively3. Multiple mechanisms are involved, including pro-inflammatory cytokines secreted by the adipose tissue, insulin resistance, and hyperinsulinism. In acute pancreatitis, obesity is a primary risk factor for developing local complications , organ failure, and death. Meta-analysis of the published studies reported a relative risk of 4.3 for local complications, 2.0 for systemic complications, and 2.1 for death4.Chronic pancreatitis is a complex disease, usually due to the exposure to several risk factors. Chronic alcohol intake was previously considered as the main environmental cause in Western countries. Since a decade ago, tobacco consumption and genetic determinism have been well established as independent risk factorset al. hypothesized that obesity before alcohol chronic pancreatitis onset might be an additional susceptibility factor5. In 227 patients with chronic alcoholic pancreatitis, 54.2% were overweight and 15% were obese before disease onset versus 37.7% and 3.1% in a control group, respectively. In this series, the highest BMI before disease onset did not impact on some major variables such as outcome of exocrine insufficiency, diabetes, calcification, and mortality from chronic pancreatitis; however, being overweight before disease onset appeared to be a risk factor for chronic pancreatitis.The link between obesity and pancreatic inflammation remains controversial. No epidemiological series have confirmed that obesity is an independent risk factor of chronic pancreatitis. However, in 2010, Ammann 7. The inflammatory mediators, including IL-6, IL-1, and TNF-α, have been implicated in the development of insulin resistance and their levels increase within adipose tissue. This inflammation is accompanied by up-regulation of anti- inflammatory factors, such as IL-10, IL-4, and TGF-β. The inflammatory mediators secreted by macrophages act not only locally, in a paracrine manner, but also contribute to systemic inflammation9. All together, these data suggest that obesity might be an independent risk factor of chronic pancreatopathy.Increasing evidence suggests that fat-derived mediators may interfere with inflammatory and immune responses. Obesity is associated with chronic low-grade inflammation , characterized by abnormal pro-inflammatory cytokine production, immune activation, disruption in autophagy, promotion of endoplasmic reticulum stress, mitochondrial dysfunction, and increased inflammatory signaling. Obesity increases the size of adipocytes, leading to their necrosis and a significant accumulation of activated macrophages14.Preliminary studies have shown the feasibility and potential diagnostic value of quantitative magnetic resonance (MR) imaging for evaluating the normal and diseased pancreas. These methods include MR elastography for assessing the degree of pancreatic fibro-inflammation, chemical shift-encoded multi-echo gradient-echo imaging for assessing the proton density fat fraction (PDFF) and transverse relaxation rate  and diffusion weighted-MR imaging with apparent diffusion coefficient (ADC) measurements for assessing pancreatic fibrosis and cellularityThe aims of this study were to characterize pancreatic endocrine and exocrine lesions in obese rats compared to a control group with pathological examinations and quantitative MR imaging and to assess the evolution of these lesions after bariatric surgery.All animal use conformed to the European Community guidelines and was approved by the local ethics committee (Comité d’Ethique Paris-Nord (no. N°121), 02285.03) and the Ministry of Higher Education and Research (N° APAFIS#8290-2016122015078962).3, n = 9) or for 6 months , with their respective control groups fed with a normal diet (ND) (Altromin 1324) for 3 months  or 6 months .Two groups of rats were fed with high-fat diet (HFD) (Altromin C1090) either for 3 months  were constituted, one operated by Roux-en-Y gastric bypass (GP)  or one the other by sleeve gastrectomy (SG) . Surgery were performed as previously described by our team and rats were sacrificed up to 3 months after the surgery15.Additionally, two surgical groups of rats fed with HFD for 3 months  and expressed in mg/dL.3-BS/GB) or SG (group HFD3-BS/SG). They were fasted overnight before operation. Anesthesia was induced by intraperitoneal injection of pentobarbital . Standard aseptic procedures were used throughout. After laparotomy, the stomach was isolated outside the abdominal cavity. Loose gastric connections to the spleen and liver were released along the greater curvature and the suspensory ligament supporting the upper fundus was severed.Male Wistar rats fed with high-fat diet for 3 months underwent BS, either GB . Then the gastric pouch was created with a TA-DST 30-mm-3.5-mm stapler , preserving the arterial and venous supply. The jejunum was transected 15 cm distally from the pylorus. The Roux limb was anastomosed to the gastric pouch and the biliopancreatic limb was anastomosed 20 cm distally to gastrojejunal anastomosis with 6-0 polydioxanone running sutures.After resection of the forestomach as described, 80% of the stomach was resected with an application of Echelon 45-mm staple gun, leaving a thin gastric tube in continuity with the esophagus and keeping the antrum in place.2. Specific stains  and immunohistochemical analyses were performed on one slide each, serially cut from this selected block.Entire pancreatic specimens were fixed in formalin and included in paraffin after sacrifice for all groups of rats. All hematoxylin-eosin–safran (HES)-stained slides of pancreas were analyzed by two investigators (AC and VR) for each case, both blinded to the group status. The number of blocks available per specimen depended on the size of the pancreas (1 or 2 blocks). We selected one block in each case for the final pathological analyses and calculated the total surface area of the pancreas for that block in cmThe HES, Picrosirius-Hemalun, and Perls stainings were evaluated by two investigators (AC and VR) blinded to both clinicopathological and outcome data of animals. Fibrosis, inflammation, acino-ductal metaplasia, fat infiltration, hemosiderin deposits and modifications of islets were independently assessed.The number of fibro-inflammatory foci per specimen were evaluated on Picrosirius-Hemalun staining. The fatty infiltration was assessed on HES staining by the count of intrapancreatic adipocytes per specimen. Acino-ductal metaplasia, assessed on HES, was defined as the replacement of acinar cells by duct cells, a switch from differentiated acinar cells to differentiated ductal cells. Hemosiderin deposits were counted on Perls stain. Islets were considered as abnormal when they were hypertrophic, hyper-vascularized, fibrotic, and disorganized or poorly delineated. These modifications were called HHF lesions. The number of HHF lesions was recorded on HES staining. For all these modifications , a ratio of the number of lesions divided by the total pancreatic surface area analyzed was calculated.Five µm sections were obtained from each selected block of pancreas. Immunohistochemistry was performed with an automated immunohistochemical stainer according to the manufacturer’s guidelines . Immunostaining was performed after the slides were dewaxed and rehydrated. Antigen retrieval was conducted by pretreatment at a high temperature at pH9 in TRIS Buffer. PBS was substituted for the primary antibody and used as a negative control. The slides were immunolabeled with monoclonal or polyclonal antibodies against insulin/β cells , CD34/endothelial cells , alpha-smooth muscle actin/activated stellate cells , CD68/macrophages , Cytokeratin 7/pancreatic ducts  and CD 45/lymphocytes  in order to analyse the islets, the microvessels, the infiltration by macrophages and lymphocytes, and the activated stellate cells.Slides stained with HES, Masson’s Trichrome and Perls were scanned with a computer-controlled capture device . We used the PRECISION image analysis software (Aperio) for the quantification of total pancreatic surfaces (HES), as well as for the quantification of fibrosis (Masson’s Trichrome), hemosiderin deposits (Perls), and islets surface (immunohistochemistry with insulin antibody) as a % of the total surface area.6, n = 6; ND6, n = 8 and HFD3-BS (high fat diet for 3 months followed by by-pass surgery), n = 5. All rats were randomly selected from their group.MR elastography and MR imaging of the entire pancreas were performed just after sacrifice for the following rats: HFD16, submerged in medium and maintained at 37 °C for the duration of the experiments. The proper positioning of the mechanical transducer was verified using a conventional T1-weighted gradient echo sequence acquired with and without a fat saturation pulse.The pancreas was dissected from the surrounding intraperitoneal adipose tissue and maintained in physiological serum. Pancreas explants were immobilized on a custom-made MR elastography actuating cradle17, relatively low frequencies of 400 Hz, 600 Hz, and 800 Hz were selected, and each frequency was assessed at 4 mechanical offsets per cycle. Acquisition parameters were TR/TE of 1000 ms/18 ms, allowing 1, 2, or 6 periods of motion encoding respectively for the three probed frequencies, 300 µm isotropic resolution, one average and a readout bandwidth of 25 kHz. Parametric maps of elasticity (storage modulus G’) and its wave dispersion coefficient or frequency dependence (γ) expressed as the exponent of a fit to a power law were obtained by algebraic inversion of the wave propagation equation after having applied the curl operator to the unwrapped shear component of the wave displacement fields18. Due to a technical failure, one pancreas in the HFD6 group could not be examined with MR elastography.MR elastography was performed using a spin-echo echoplanar sequence with motion encoding gradients used sequentially along the three directions. To accommodate the softness of pancreatic tissue21.A chemical shift-encoded gradient-echo MR imaging sequence with 15 consecutive echoes  was used to calculate the pancreatic fat fraction and R2*. The other parameters were in-plane resolution of 250 µm, 1 mm slice thickness, 300 kHz bandwidth, 950 ms TR, 15° flip angle, and 4 signal averages. Parametric maps of fat fraction and R2* were extracted by fitting the magnitude images to a model accounting for transverse relaxation time (T2*) decay, noise, and signal interferences between water and the seven most abundant lipid peaksex vivo explants, no perfusion component was expected, hence the fitted equation was S(bi) = S0 × exp(−ADC × bi), with S(bi) the signal obtained at the i-th b value, S0 the signal in absence of diffusion weighting, ADC the apparent diffusion coefficient and bi the various b values. ADC and S0 were kept as free parameters.Diffusion-weighted MR imaging was performed with a spin-echo sequence and 21 segment echoplanar readout, 4 b-values , 195 µm in-plane resolution, 400 µm slice thickness, 2000 ms/20 ms TR/TE, a readout bandwidth of 250 kHz and 3 signal averages. The apparent diffusion coefficient (ADC) was calculated on a pixel-by-pixel basis using custom made software running the root framework . As the images concerned Regions of interest were selected on the MR elastography maps. The regions of interest were positioned at the center of the pancreas, while avoiding the MR elastography piston transducer. The extent of the regions of interest was maximized under the constraint of having mechanical wave amplitudes larger than 8 µm. This step was taken to ensure accuracy of the MR elastography results by providing sufficiently strong wave amplitudes for proper inversion of the wave equation. Regions of interest defined with this procedure were then reported onto the other parametric maps with appropriate geometric transformations to accommodate the various fields of view and resolutions.General characteristics were expressed as medians and ranges, or percentages. Comparisons of general and morphological characteristics, MR, and pathological data between groups were performed using the Mann-Whitney test or the Kruskall–Wallis test and Dunn post-test for continuous data and with the Chi2 test or the Fisher’s exact test for categorical data. Spearman correlation coefficients were used to search for correlations between continuous variables.http://www.medcalc.org; 2017). All statistical tests were two-sided. The critical level of statistical significance was set at p < 0.05.Data were analyzed with the SAS 9.1 statistical software for Windows  and MedCalc Statistical Software version 17.6 , P = 0.0005. Older rats presented more HHF lesions, P = 0.0139. Foci of fibrosis with acino-ductal metaplasia lesions were quite significantly more found in HFD6, P = 0.0843. The median glucose blood level was higher in the group HFD6, P = 0.0063. Adipocyte infiltration (P = 0.136) and iron deposits (P = 0.23) did not significantly differ between the two groups. Figure The median weight before sacrifice was higher in the group No significant difference was found between the 2 groups regarding the number of fibrosis foci, iron deposits, acino-ductal metaplasia lesions, adipocytes (fat infiltration), and HHF complexes. The weight before sacrifice was not significantly different.The comparison between HFD and ND rats showed an increase of HHF lesions (P < 0.0001), hemosiderin deposits (P < 0.0001), adipocyte infiltration (P = 0.0008), and acino-ductal metaplasia lesions and fibrosis (P = 0.0424) in HFD rats. The increase of the weight in HFD rats was associated adipocyte infiltration (P = 0.009). In the 4 groups, HHF lesions were associated with hemosiderin deposits . Figure In the surgery group, no difference was found regarding morphological lesions according to the type of procedures (by pass or sleeve gastrectomy).6 and HFD-bariatric surgery groups, operated rats presented less HHF lesions (P = 0.0004), fat infiltrate (P = 0.005), and acino-ductal metaplasia lesions (P = 0.05). Figures An analysis was performed in HFD rats after bariatric surgery compared to HFD rats fed during 3 months. In rats operated on, the number of adipocytes (P = 0.017) drastically decreased. However, HHF, acino-ductal metaplasia and iron deposits were not different between the 2 groups. When the analyses were conducted between the HFD6) weighing 863 ± 141 g, the control group (ND6) weighing 586 ± 42 g, and the bariatric surgery group weighing 640 g [508–680].Imaging procedures were performed in rats from the obese group . Post-hoc tests showed that the storage modulus was significantly higher in obese rats versus controls (P < 0.01). After surgery the elasticity tended to decrease non-significantly to a value that was not significantly different from the value in controls. Figure The wave dispersion coefficient was significantly different between the three groups (p = 0.02). Values were significantly lower in the obese group than in the control group (post-hoc P < 0.05). In the surgical group (1.55 [1.15–1.65] kPa), the wave dispersion coefficient had a value close to that in the control group, without significant differences relative to the control and high fat diet groups. Figure When considering the histology results, the elasticity was positively correlated to the number of fibro-inflammatory complexes  Fig. . The wavOverall, the fat content measured at MR imaging from both groups was low. The obese group had a significantly higher pancreatic fat content than the control group . The pancreatic fat content of the surgery group was intermediate (0.20 [0.00–0.49]%) and statistically indistinguishable from either the control or the obese groups. Pancreatic fat content determined with MR imaging was highly correlated to the number of adipocytes per unit area , while the surgery group had R2* values of 83.80 [64.57–90.20], close to those observed in the control group. Values in the obese group were significantly higher than those obtained in the surgery (P < 0.001) and in the control (P < 0.001) groups, while values in the surgery group were not significantly different from those in the control group. A significant correlation was observed between R2* and the number of iron deposits   Fig. .The ADC was not significantly different between any group with values of 0.73 [0.57–0.79], 0.74 [0.63–0.95] and 0.73 [0.71–0.77], for the control, obese and surgery groups, respectively. No differences were found between animals displaying or not displaying intralobular adipocyte infiltration on pathological slides. No significant correlation was found between the ADC and the number of HHF.Representative images of a pancreas explant at MRI are showed in Fig. In this study, we assessed pancreatic lesions associated with obesity and high fat diet. Obese rats developed adipocyte infiltration and exocrine lesions of pancreatopathy characterized by acinar-ductal metaplasia, fibrosis, and iron deposits mainly located at the endocrine-exocrine interface. These lesions increased with the animal weight and duration of the diet, and could be assessed by MR imaging and elastography and partly reversed after bariatric surgery.22. Even if the pathways between obesity and inflammation are not fully understood, several mechanisms have been proposed. The expansion of fat mass, due to adipocyte enlargement, increases the fat infiltration by macrophages, and the inflammatory M1-type, especially23. Obesity promotes macrophage recruitment and the secretion of numerous inflammation cytokines as TNFβ, IL-1, IL-6, and IL-18. These inflammatory mediators promote a chronic systemic inflammation in a paracrine manner. This study confirmed a direct link between pancreatic pathological changes and obesity. However the respective roles of the fat tissue, the high fat diet and the pancreatic fat infiltration in the pancreatic toxicity can be addressed to explain these changes on endocrine and exocrine tissue.Obesity is the most common cause of insulin resistance and increases susceptibility for pancreatic disorders, such as pancreatitis and pancreatic adenocarcinoma, by involving low- grade chronic inflammation24. In vitro studies and mice models using high fat diets showed that lipid peroxidation products play a key role in stellate cell activation and fibrogenesis. Moreover, Philip et al. showed that HFD can activate oncogenic Kras via COX2, leading to pancreatic inflammation and fibrosis, and development of PanINs and pancreatic cancer in mice. Consumption of a HFD can be an inflammatory stimulus to trigger Kras signalling, initiating the development of cancer25.Animal models have shown that chronic high fat diets in rats induce fibrogenesis26. However, the crosstalk between adipocytes, pancreatic stellate cells, endocrine, and exocrine tissue is still unknown.The implications of pancreatic fat with reference to its proximity to pancreatic acinar cells and its toxic effects on acinar cells are still unknown. Fat adjacent to acinar tissue has been shown to be associated with the worst parenchymal damages immediately around the fat in acute pancreatitis in case of adipocyte necrosis27.In this study, fibrosis and acinar-ductal metaplasia were more prevalent in obese rats. ADM is first a part of regenerative process in the pancreas but in long term ADM is also considered as the first step of pancreatic oncogenesis. It is possible that these long-term changes modify the pancreatic environment with potential cancer risk. The main interest of this work is to show that these lesions could regress after surgery. Regarding the over risk of pancreatic cancer in obese patients, this point has to be specifically highlighted. As already published, this phenotype change is promoted by inflammation and pancreatic injuries. Our results are in keeping with the finding that obesity and pancreatic fatty infiltration are risk factors of PanIN lesions in humans. We previously reported that fatty infiltration, especially in intra lobular location, could promote precancerous lesions (OR = 17.86 [4.935–88.12]), independently of the age of the patients and the presence of diabetes29. Hence, it is assumed that the high fat-induced peripheral insulin resistance could promote a compensatory islet cell proliferation, followed by islet inflammation-mediated degeneration and fibrosis30. In a previous study, Schneider et al. showed that HFD induced an altered local insulin release and was associated with a significant increase in islet cell turnover and pancreatic cancer induction in rodents31. In our model, we did not find differences in insulin staining in b-cells in HFD rats and could not confirm the hypothesis discussed in recent mice models of diet-induced obesity32. However, as in the literature, we found an increase of the oral glucose tolerance test and insulin secretion after bariatric surgery34. Interestingly, we demonstrated that in our model, abnormal pancreatic features were detected not only in endocrine, but also in the exocrine pancreas, mainly observed beside HHF lesions, consisting in acinar-ductal metaplasia with inflammation, fibrosis, activation of stellate cells and iron deposits. Chronic inflammation and early precancerous lesions, locally favoured by both HHF and fatty infiltration, could account for higher risk of cancer in obese patients. Fibrosis and hypervascularization may induce local micro haemorrhage and iron overload that may amplify pancreatic damages.The histopathological studies of the pancreas of obese rats showed numerous and severe lesions involving islets and their surrounding exocrine parenchyma, that we called HHF, combining hypertrophy, hyper-vascularization and islets fibrosis. These features were correlated with glycoregulation abnormalities and increases in the rat weight. So both islet morphology and function are altered in obese rats. These islets characteristics have been partly observed in other models of obesity such as Zucker fatty rats or WNIN mutant rats32. No experimental data reported morphological changes of pancreatic lesions after surgery. Our results showed for the first time the impact of bariatric surgery on obesity-induced pancreatopathy, which is relevant for humans. In our series, no difference was found regarding morphological lesions according to the type of procedures (by pass or sleeve gastrectomy).The originality of our model is the evaluation of bariatric surgery as a potential treatment and prevention of fibrosis and precancerous lesions . Lesions were indeed reversible. While bariatric surgery is one of the more efficient treatments of morbid obesity, few data are available in the literature to assess its consequences on systemic inflammatory profiles. It is also difficult to determine the precise effect of different bariatric surgical procedures on the beta cell function, particularly the early insulin secretion after stimulation by glucose, because of the considerable heterogeneity among studies35. Recently, it has been reported that the wave scattering coefficient and the related “damping ratio” parameter at MR elastography are complementary markers of liver inflammation37.In this study, the pathological changes could be assessed with quantitative MR imaging. We observed significant changes of pancreatic mechanical properties, proton density fat fraction and R2* in obese rats. These changes paralleled the increases in HHF, adipocytes and iron deposits observed with obesity. Moreover, the changes in the MR and pathological features reversed significantly in parallel after surgery. Particularly, significant correlations were observed between pancreatic elasticity and wave scattering coefficient at MR elastography on one hand and the number of fibrotic lesions at pathology on the other. These results are in agreement with reported findings. Liver elasticity measurements at MR elastography are accurate markers of liver fibrosis stage in patients and animal models38. Such findings have not yet been reported with MR elastography but the feasibility and reproducibility for pancreatic MR elastography were reported in patients40.Few elastography studies have been performed in the pancreas, mostly with ultrasound elastography. Increased stiffness values related to fibrosis and inflammation have been reported with ultrasound elastography in chronic and acute pancreatitis in clinical studies and in one mice study41. Proton density fat fraction has been used to assess pancreatic fat in clinical studies14. As shown in our study, it is important to use T2* corrected MR sequences to assess PDFF in the pancreas because iron deposits (and the related R2* measurements) increase in obese rats42.We observed significant changes of proton density fat fraction and R2* at MR elastography in obese rats; these MR parameters were correlated to pancreatic fat and iron spots at histopathology. Obesity is associated with ectopic fat deposition in parenchyma organs such as pancreaset al. who observed a lack of ADC change related to pancreatic fibrosis in patients who had MR Imaging before surgical pancreatectomy14.We did not observe significant changes in ADC in obese rats versus controls. These results are in agreement with the findings of Yoon JH ex vivo. The absence of perfusion in our model may have influenced the results of MR elastography and diffusion-weighted MR imaging43. The results have thus to be confirmed in vivo in future studies. Other MR sequences have also been proposed to stage pancreatic fibrosis, including intravoxel incoherent motion diffusion-weighted MR imaging14, kurtosis diffusion-weighted MR imaging44 and T1 relaxometry45. However, in the liver, these MR methods have not yet been shown to be more accurate than MR elastography to stage fibrosis46.Our MR imaging study was performed ex vivo MR imaging study suggest that the reversible changes in pancreatic fat, iron, and fibrosis in obese rats can be quantified with quantitative MR imaging using multi-echo gradient echo MR imaging and three-dimensional MR elastography. Applications in humans have to be developed in order to propose a new screening tool, especially to detect obese patients at high risk of pancreatic cancer.Several limitations could be addressed and the descriptive characteristics of the results especially. No mechanistic insight is proposed to understand the physiopathological processes. However, we confirmed that high fat diet induced obesity is a risk factor for pancreatopathy and inflammatory pancreatic lesions especially, both in acinar location and at the endocrine-exocrine interface and for early precancerous lesions. Bariatric surgery and obesity management can reverse these lesions. The results of our


This study aimed to investigate the efficacy of percutaneous hemiepiphysiodesis for gradual correction of symptomatic juvenile hallux valgus (HV) deformity.Between 2012 to 2014, 24 patients with symptomatic juvenile HV were treated by combined percutaneous medial drilling hemiepiphysiodesis of the first proximal phalanx and lateral transphyseal screw hemiepiphysiodesis of the first metatarsal at our institution. Twenty-one of 24 patients fulfilled inclusion criteria had a complete radiological and clinical follow-up of at least 2 years. Preoperative and postoperative radiographs of the feet were reviewed for measurements of hallux valgus angle (HVA), intermetatarsal angle (IMA), proximal metatarsal articular angle (PMAA), proximal phalangeal articular angle (PPAA), and metatarsal length ratio (MTLR). Clinical outcomes were assessed using the AOFAS hallux metatarsophalangeal-interphalangeal score.P < .001) and the reduction of IMA by 2.2 degrees (P < .001). The PMAA and PPAA also improved significantly in the anteroposterior plane; however, the PMAA difference was insignificant in lateral plane as expected. The mean difference in the MTLR was 0.00 (P = .216) which was indicative of no length discrepancy between first and second metatarsals. The AOFAS score increased from 68.7 to 85.2 (P < .001). In correlation analysis, time to physeal closure was significantly correlated with the final HVA change .The study included 21 consecutive patients (37 ft) for analysis. The mean age at surgery was 12.0 years (SD = 1.3) and mean follow-up after surgery was 35.1 months (SD = 6.0). With the data available, the HV deformity improved in terms of the reduction of HVA by a mean of 4.7 degrees (Although combined hemiepiphysiodesis does not create a large degree of correction as osteotomy, yet it did improve HV deformity with adequate growth remaining in our series. It is a procedure that can be of benefit to patients with symptomatic juvenile HV from this minimal operative approach before skeletal maturity.Level IV, retrospective case series. Juvenile or adolescent hallux valgus (HV), also known as bunions, is a forefoot deformity commonly seen in the skeletally immature population , 2. The The management of HV deformities in skeletally immature patients remain controversial, either conservative or operative treatments. With uncertainty of halting progression in conservative management, operative intervention is often required in symptomatic skeletally immature HV , 4. HoweThe hemiepiphysiodesis in skeletally immature patients, by tethering marginal physis and creating asymmetrical physeal growth, has been used for treatment of angular deformities of the lower limbs for many years . The potWe hypothesize that combined hemiepiphysiodesis targeting the first ray proximal phalangeal and metatarsal physis is effective in management of juvenile HV with adequate growth remaining. The aims are to investigate the efficacy and the complication associated with the combined procedures and the factors related to the operative outcomes.We retrospectively reviewed the case series of all patients with juvenile HV (HV angle >16 degrees) who received combined hemiepiphysiodesis surgery from 2012 to 2014 at our institution. Pre-operatively, all included patients complained of pain, redness, or callosity at the bunion with failed conservative treatment, including analgesics, shoe modification or orthotics. Other requirements for inclusion were patients with a minimum of 2 years of complete radiographic and clinical follow-up. Patients with neuromuscular disease, juvenile rheumatoid arthritis and connective tissue disorders were eliminated. The medical records were reviewed for chief complaints, age at surgery, length of follow-up, the need for future foot surgeries, and complications. The study has been approved by the Research Ethics Committee of National Taiwan University Hospital (201601015RIND).All radiographs of the feet were taken in standard weight-bearing anterior-posterior (AP) and lateral view of foot before surgery and at 3 to 6-month interval postoperatively until the latest follow-up. All patients had their physis closed at final follow up and the time from surgery to radiographic physeal closure was recorded. We measured the hallux valgus angle (HVA), intermetatarsal angle (IMA), proximal metatarsal articular angle (PMAA), proximal phalangeal articular angle (PPAA) Fig. a & b, thFor the reliability test of radiographic measuring, Intraclass Correlation Coefficient (ICC) was used to analyze the intra-rater and inter-rater reliabilities. Regarding intra-rater reliability, the measurement of above parameters was repeated at 1-week interval by a junior author using AGFA-Orthopaedic-Tools Version 2.10 . For inter-rater reliability, two authors measured the above parameters independently using the same software.The functional assessments were evaluated with the American Orthopaedic Foot & Ankle Society (AOFAS) hallux metatarsophalangeal-interphalangeal score which comprised of nine questions and cover three categories: Pain (40 points), function (50 points) and alignment (10 points). These are all scored together for a total of 0 to 100 points . All funAll procedures were carried out identically by the two senior authors who worked as a team. With patient on supine position under general anesthesia and fluoroscopic control, a guided wire for the cannulated screw is positioned retrograde from the medial cortex of first metatarsal mid-shaft and pointed toward the lateral and proximal corner of first metatarsus. The guided wire is ideally crossed through the lateral quarter of first metatarsal physis in AP view and centered of physis in lateral view Fig. . After gp value of < 0.05 was defined to be statistically significant.All of the parameters were checked for normality first using the Shapiro-Wilk test. Descriptive statistics were given as mean ± standard deviation (SD). The paired sample t-test was used for comparison of the preoperative and the latest radiographic results following index surgery. Pearson’s correlation test was used to analyze the relationship between the final change of HVA and preoperative demographic variables including age at surgery, time to physeal closure and radiographic parameters. All statistical analyses were performed using SPSS software, version 17.0 , and a During the period, 43 ft of 24 patients had index procedures for symptomatic juvenile HV. Four feet from 2 patients with cerebral palsy were excluded. There was one patient who did not return for 2 years follow-up. A total of 37 ft in 21 patients (9 boys and 12 girls) met our inclusion criteria for subsequent analysis. The mean age at surgery was 12.0 (SD = 1.3) years. The mean age of the girls and boys at time of surgery were 11.2 (SD = 0.9) years and 13.0 (SD = 1.1) years respectively. Mean follow-up after surgery was 35.1 (SD = 6.0) months. The mean time period from surgery to physeal closure radiographically was 15 (SD = 4.8) months. The average screw position-AP was 0.25 (SD = 0.07) which indicated the position of all screws crossing was consistently located at lateral quarter of the first metatarsal physis in the coronal plane. The screw position-LAT was 0.56 (SD = .07). The demographic data were summarized in Table P < .001). The HVA improved in 33 of 37 ft. Four feet in 4 patients had slight progression of HVA at final follow-up. The IMA of treated feet also significantly improved by a mean of 2.2 degrees (P < .001). These feet had a mean preoperative IMA of 12.3 degrees and reduced to a mean final IMA of 10.0 degrees. The IMA improved in 32 of 37 ft. The mean correction of PMAA-AP and PPAA were 2.5 degrees (P = .004) and 1.9 degrees (P < .001), respectively. These feet had a mean preoperative PMAA-AP of 91.9 degrees and decreased to final of 89.4 degrees. The mean change of the metatarsal sagittal alignment (PMAA-LAT) was 0.2 degrees upward without statistical significance (P = .564). The mean change in the MTLR was too small without statistical significance (P = .216). The ICC for intra- and inter-rater reliability in all radiographic measurements was greater than 0.80. .Several preoperative clinical factors were analyzed with correlation test to identify potential relationships with the amount of final HVA change. The results are summarized in Table P < .001). All patients had rapid return to school and sports activities. There was no known complication or overcorrection. No patients required revision surgery for residual HV after index procedure.At the final visit, the AOFAS hallux metatarsophalangeal-interphalangeal scores significantly increased from 68.7 to 85.2 ; therefore, less threads can purchase epiphysis. The failure of other 3 ft might be attributed to late timing for surgery . From our analysis, time to physeal closure was correlated with the final HVA change. Although there is still no consensus on the optimal timing of metatarsal hemiepiphysiodesis, previous studies suggested that adequate growth remaining to achieve significant correction by hemiepiphysiodesis is up to 10 years for girls and up to 12 years for boys . The meaThis study had two limitations. First, this is a retrospective case series study with limited sample size. Second, this study didn’t directly compare with untreated patients with juvenile HV. In spite of this, most of the patients in this study had improvement in HVA and IMA, which were better than those following their natural history .With adequate growth remaining, the combined metatarsal and proximal phalangeal hemiepiphysiodesis is a simple and effective procedure for juvenile HV patients with minimal approach. Although combined hemiepiphysiodesis does not create a large degree of correction as osteotomy, the percutaneous procedures greatly reduce the postoperative disability with immediate weight-bearing. The metatarsal length ratio and the sagittal alignment of the first metatarsal were retained. Longer follow-up is needed to observe possible recurrence of the deformity. In the future, a head-to-head comparison with non-surgical cases would help to draw firm conclusions.


As an epigenetic player, long noncoding RNAs (LncRNAs) have been reported to participate in multiple biological processes; however, their biological functions in silica-induced pulmonary fibrosis (SIPF) occurrence and development remain incompletely understood. Five case/control pairs were used to perform integrated transcriptomes analysis of lncRNA and mRNA. Prediction of lncRNA and mRNA functions was aided by the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Additionally, we constructed a coexpression network of lncRNAs and mRNAs to identify targets of regulation. In total, 1069 differentially expressed mRNAs and 366 lncRNAs were identified with the changes more than 2 times (p<0.05), of which 351 downregulated mRNA and 31 downregulated lncRNA were <0.5 (p<0.05) and those of 718 upregulated mRNAs and 335 upregulated lncRNA were >2 (p<0.05). The levels of 10 lncRNAs were measured via qRT-PCR; the results were consistent with the microarray data. Four genes named of FEM1B, TRIM39, TRIM32, and KLHL15 were enriched significantly with ubiquitination and immune response. Cytokine-cytokine receptor interaction was the most significantly enriched KEGG pathway in both mRNAs and lncRNAs. The coexpression network revealed that a single lncRNA can interact with multiple mRNAs, and vice versa. lncRNA and mRNA expression were aberrant in patients with SIPF compared to controls, indicating that differentially expressed lncRNAs and mRNAs may play critical roles in SIPF development. Our study affords new insights into the molecular mechanisms of SIPF and identifies potential biomarkers and targets for SIPF diagnosis and treatment. Evidence has been presented that silica dust acute and chronic exposures will trigger an inflammatory cascade, such as macrophages and alveolar epithelial cells proliferated successively, followed by inflammatory cascade; the fibrotic development will be ignited, which eventually will proceed to determine the silicosis , 2. Siliβ1 synthesis  . HO-1, tfibrosis ). MiR-44fibrosis . miR-19afibrosis . The miRfibrosis . lncRNA fibrosis  modulatifibrosis . lncRNA-fibrosis . Thus, t3, exceeding the exposure limit of 0.2–1 mg/m3 (GBZ2-2002). At least 5 mL of peripheral blood was collected from each subject into a PAX gene Blood RNA tube , held at room temperature (22–25°C) for at least 2 h to completely lyse all cells, placed at –70°C and immediately delivered to the Beijing Kangpusen Biotech Company for microarray analysis. The study was performed in accordance with all relevant tenets of the Declaration of Helsinki and was approved by the Ethics Committee of Xiangya School of Public Health . All participants provided written informed consent.We enrolled 10 subjects: 5 with phase I lung fibrosis (cases) and 5 healthy subjects who worked in the same industry but were not exposed to silica (controls). The inclusion criteria were (1) an occupational history of silica exposure and a fibrosis diagnosis based on the Chinese Pneumoconiosis Diagnosis Standards (GBZ70-2013), as agreed by at least three occupational physicians; (2) lack of complications such as tuberculosis, lung cancer, or an infection; and (3) voluntary agreement to participate. The mean age of the cases was 55±6.2 years and that of controls was 51±7.9 years; all participants were male. The silica dust concentration in the workplace was 6.43±0.77 mg/mμL of miRNA wash solution and 500 μL general wash solution; then, RNA was eluted into 100 μL of elution solution at 95°C and stored at –70°C until analyzed. RNA purity and concentration were assessed using a QIAGEN RNeasy Mini Kit and by deriving a spectrophotometric ratio with the aid of a NanoDrop-2000 instrument, respectively .Total RNA was isolated using the mirVanaRNA Isolation Kit following the manufacturer's instructions. Briefly, a 1.25 volume of absolute ethanol was added to each blood sample followed by mixing; the mixture was passed through a filter and the filter was washed three times with 700 μL of first-strand buffer mix and 1 μL of first-strand enzyme solution were then mixed and added, and first-strand was cDNA synthesized; this was followed by second-strand synthesis. Purified cDNA and second-cycle single-strand cDNA were subjected to WT cartridge hybridization, washed, and scanned using a DNA Microarray Scanner . Expression Console software (ver. 1.4.1) was used to adjust the raw data background and standardize microarray data.In brief, microarray analysis was performed as follows: RNA/-poly-A-RNA-control mixtures were prepared by mixing test RNA samples (50–500 ng), a diluted poly-A-RNA control, and nuclease-free water; 4 Transcriptome Analysis Console  software was employed to explore the differential expression of mRNAs and lncRNAs between cases and controls; we selected RNAs exhibiting the greatest fold differences with reference to the p values. Cluster analysis  was usedhttp://www.geneontology.org). The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was employed for pathway enrichment analysis.Gene Ontology (GO) and pathway enrichment analysis were performed to predict biological processes, cellular components, and molecular functions affected in disease. We used a controlled gene vocabulary  and generated visual data. We included five essential lncRNAs and all essential mRNAs in a network assessment. Only differentially expressed mRNAs with Pearson correlation coefficients ≥ 0.90 (p<0.01) were used to construct the lncRNA-mRNA network and generate visual representations.Δ  Δ  CT method. The primer sequences are shown in Total RNA was isolated and reverse-transcribed into cDNA using a PEXBIO RNA kit according to the manufacturer's protocol. qPCR was performed with the aid of Super Real PreMix Plus  on a CFX96TM Real-Time System . GAPDH served as the internal control and fold change calculations were made using the 2–2 transformation. The fold changes between cases and controls were calculated; changes >2 or <0.5 were considered to indicate differential expression; p<0.05 was taken to indicate significance.R software  was used to compare the expression levels of mRNAs and lncRNAs, for cluster analysis, and to draw volcano maps. One-way ANOVA was employed to explore the significance of differences in mRNA and lncRNA levels after logp<0.05), of which one-way ANOVA showed that the fold changes of 351 downregulated mRNA were < 0.5 (p<0.05) and those of 718 upregulated mRNAs were > 2 (p<0.05). The most prominently upregulated mRNA was TC0700007850.hg.1 . The top 25 up- and downregulated mRNAs are listed in p<0.05), of which one-way ANOVA showed that the fold changes of 31 downregulated lncRNAs were <0.5 (p<0.05) and those of 335 upregulated lncRNAs were >2 (p<0.05). The most prominently downregulated lncRNA was TC1300009322.hg.1, with a fold change of 0.395 (p<0.05). The most prominently upregulated lncRNA was TC1700010761.hg.1, with a fold change of 6.70 (p<0.05). The top 25 up- and downregulated lncRNAs are listed in Through microarray analysis, 1069 differentially expressed mRNAs were identified with the changes more than 2 times  along with more lncRNAs were up- than downregulated  that determined the essential differences between cases and controls Figures . These lny mRNAs .Microarray and bioinformatics techniques now allow us to detect changes in lncRNA expression; most work to date has focused on miRNAs –33. lncR LIMS2, ASB1, and HHAT were upregulated and those encoding TRANK1, IFIT1, and SLC15A3 were downregulated [ CTFR expression was regulated at the mRNA level [ LOYBOY, DEEZOY, and SNEYSPORBY were upregulated, and those encoding CPA3, FCER1A, and CCR5 were downregulated; these mRNAs may be involved in silica-induced lung fibrosis. Although lncRNAs may be potential therapeutic targets in fibrosis [Differentially expressed mRNAs and lncRNAs have been reported to be associated with development of lung fibrosis. Laurent et al. explored the cultured lung fibroblast transcriptome of idiopathic pulmonary fibrosis; mRNAs encodingegulated . Biswas NA level . CertainNA level , 40. We fibrosis , the onlfibrosis . Our stuGo and KEGG analyses are commonly used to predict biological functions and/or signaling pathways affected by mRNAs and lncRNAs. On GO analysis of significant changes in mRNAs , in terms of biological proses, cellular components, and molecular function, the most-affected processes were interleukin-12 secretion, regulation of intracellular mRNA localization, effects on the dimeric IgA immunoglobulin complex, C-C chemokine binding, inflammation response, immune response, ubiquitin-like protein-specific protease activity, and thiol-dependent ubiquitinyl hydrolase activity. Here, the results which the inflammation response affected by mRNAs are consistent with the previous report; however, mRNAs affecting the ubiquitination and immune response are new insights into the mRNAs function in the lung fibrosis. This suggests mRNAs might participate in lung fibrosis process by regulating immune response and ubiquitination. Ubiquitin is a highly conserved low-molecular-weight protein and ubiquitination represents a very common posttranslational modification. Extracellular ubiquitin regulates the immune response and exhibits anti-inflammatory and neuroprotective activities. Zhou et al. reported that silica activated macrophages and increased circRNA HECTD1 levels via ubiquitination . Tsuboucα and exogenous dsRNA. The KEGG data showed that the lncRNA participated in multiple pathways, including the herpes simplex infection pathway, a hepatitis C pathway involving inflammation, and the PPAR signaling pathway  were consistent with the microarray data. Our results suggest that (1) silica exposure modulates the expression of both lncRNAs and genes and (2) some silica-induced genes may be involved in ubiquitination, and some lncRNAs may affect immune cell interactions. The mechanisms by which lncRNA and mRNA coexpression mediates SIPF require further study.However, our study had certain limitations. First, the lncRNAs measured via qRT-PCR constituted only a small proportion of those identified via microarray; some lncRNAs may have been missed such that further studies are required. Second, we recruited only five cases; studies with larger samples are required. However, we are the first to use human peripheral blood samples to identify lncRNAs and mRNAs in patients with SIPF.Gene and lncRNA expression profiles were dysregulated in the peripheral blood of SIPF patients, suggesting future diagnostic and therapeutic biomarkers. LncRNA dysregulation may play an important role in disease development, given its plethora of effects on posttranscriptional mRNA regulation. However, further biology function work is required.