Blending polyolefins with certain types of natural polymers like starch can be beneficial to their biodegradation. The impact of alpha-amylase on the biodegradation of low-density polyethylene (LDPE)-starch blend samples in an aqueous solution was investigated through characterizing their physical, mechanical and chemical properties. Results indicated that the weight and tensile strength of the enzyme treated samples were reduced by 48% and 87% respectively. Moreover, differential scanning calorimetry (DSC) showed an increase in fusion enthalpy of degraded samples which means that the crystallinity has been increased. The biodegradation of LLDPE appeared in Fourier-transform infrared spectroscopy (FT-IR) through the reduction in the intensity of the related peaks. This observation was supported by energy dispersive x-ray spectroscopy (EDXS) analysis where decreasing the percentage of carbon atoms in the treated blend was obtained. Likewise, the gel permeation chromatography (GPC) results pointed to a significant reduction in both the molecular weight and viscosity of LDPE more than 70% and 60% respectively. Furthermore, thermal gravimetric analysis (TGA) affirmed the function of amylase in degradation of the blend. On the basis of the obtained results, it can be claimed that the main backbone of the polymer, as well as the side branches, have been scissored by the enzyme activity. In other words, alpha-amylase has a promiscuous cometabolic effect on biodegradation of LDPE in polymer-starch blends. In addition, it has been reported that blending of the polyolefins with certain types of natural polymers such as the starch can be beneficial to their biodegradation5. The specific characteristics of the starch in combination with the biodegradability lead to be considered as an alternative to the petroleum-based plastics in the form of composites and blends with the proper materials6. In fact, owing to the properties such as the gel formation, adhesion, swelling, physical and chemical transformation capabilities, starch has been suggested as a principal part of several polymeric mixtures to find applications in the medical, food and scientific domains8.Polyolefins have been extensively utilized in a wide range of industrial and domestic applications. They have many advantages such as suitable chemical and physical properties, low prices, high capacity of production, and well-established facilities and processing methods which support their increasing usage trend in the coming years. However, the recalcitrant nature of these petroleum-based polymers makes them a threat to the environment. Although the biodegradation of polyolefins is feasible in the natural environment, it can prolong to a few centuries. As a remedy, several solutions such as photodegradation, thermal degradation, and addition of degradation accelerators  have been suggested to enhance their biodegradability9. The suggested mechanism for the biodegradation improvement has been related to the fast hydrolysis of starch which makes the polymer porous and more susceptible to both biotic and abiotic degradations12. Consequently, the changes in the physical structure and crystallinity of the polymer will affect its mechanical properties13. Furthermore, it has been reported that starch has a synergetic effect on UV photo-oxidative degradation of PE through enhancing the production of carbonyl groups in the polymer structure12. However, reports on biodegradation of PE-starch blends indicated that with the exception of the presence of pro-oxidants in the system, the molar mass of polyethylene was nearly unchanged even though the physical and mechanical properties of the samples have decreased significantly17. Although the presented evidence in the literature supports the suggested mechanisms, the above explanations for polyolefins biodegradation improvement seem to be incomplete18. It has been reported that the accessibility of starch in its blend with a polymer has been greatly limited especially in lower starch contributions  which are quite usual in preparing the blends19. In fact, most of the presented scanning electron microscopy (SEM) pictures of the starch polymer blends after the degradation process have shown a few superficial tiny cracks and pores which cannot affect the available surface area of the polymer significantly12. A fine powder of the polymer would be more susceptible to degradation if the surface area were helpful for PE degradation. On the other hand, considering the production of active moieties and free radicals as the driving force of degradation is hardly satisfactory because the free radicals react through a fast propagating mechanism and the polymer should have demolished in a short time20.The weight and mechanical properties of the starch blended polymers were reported to be lower than the blank samples after treatment with microorganisms21. Accordingly, the similarity between the chemical structure of paraffin hydrocarbons and polyethylene can inspire that the biodegradation of polyethylene is likely to be enhanced in the presence of amylase in the same way. In fact, the possible chemical decomposition of the polyethylene molecules at the presence of starch and amylase can lead to decrease in the molecular weight of the polymer. Besides, a blend of starch with a hydrophobic polymer like PE can be stabilized through the inclusion mechanism with the V-type conformation of the starch molecules24. The partial decomposition of starch in such a blend can unbalance the hydrophobic-hydrophilic interactions which cause cracks in the physical structure of the system. In this work, an attempt is made to investigate the above-mentioned mechanisms for degradation of a starch-PE blend in the presence of alpha-amylase.In fact, the primary target of blending PE with starch is elevating the degradation of polyethylene in the prone conditions. The starch is well-known as a biodegradable polymer and in almost all of the published researches its degradation in the blend has been reported. Therefore, it is rational to think that there must be something about the degradation of starch affecting the PE breakdown. Alpha-amylase is a specific enzyme for starch hydrolysis, and the starch biodegradation has been mainly referred to its activity. Consequently, it can be considered as an effective factor in the PE degradation. Recently, it has been reported that amylase can be effective on the degradation of saturated linear hydrocarbons25. The observed biodegradation here has been studied in more details in the following sections.The Physical changes in the body of samples can be considered as the first clue of degradation. In fact, decomposition of materials in a degradation process accompanies with apparent deficiencies in the shape of the sample. The physical appearance of the blank and treated samples has been shown in Fig. 27. The A-type starch can be converted to the V-type through plasticization by a plasticizer and implementation of shear force in an extruding process6. Previous studies which have reported scanning electron micrographs for the native starch shown spherical and oval granules in various sizes, without craze line, crack, pores and fissure while the thermoplastic starch micrographs shown a smooth surface29. It has been suggested that in the plasticization of starch, H-bonds’ failure between the starch molecules and the formation of H-bonds between starch and plasticizer molecules (e.g. glycerol) can simultaneously occur30. Moreover, an increase in the percentage of glycerol leads to increasing the average intermolecular distance by swelling31. Therefore, the plasticization effect obtained by both the addition of glycerol (as the plasticizer) and the shear forces in the samples producing procedure in this work reduces the interactions between the starch chains and simplifies their movement32. The homogenous surface without any granules which has been taken by scanning electron microscopy (SEM)  supports the idea that the starch has been formed in the V-type structure in the blend with PE. Moreover, the partial hydrolysis of the starch in the blend with PE due to the effect of amylase has caused loss of the sample integrity by the formation of some physical defects such as cavities, pinholes, and non-level multiple layers. Fig. α) displays the surface of a PE-starch sample subjected to the enzymatic degradation with the mentioned defects. Several more micrographs of the samples have been shown in Fig. It is reported that the native starch has an A-type semi-crystalline structure while the thermoplastic starch forms the V-type crystalline conformationα and Bs) and untreated blended (A0) samples while a significant reduction was detected in PE-starch blend treated by the enzyme (Aα). Interestingly, the weight reduction percent in the presence of alpha-amylase was more than the initial starch amount (25%) in each sample. This means that in addition to starch, polyethylene has been degraded in the system. In fact, there should be a cometabolic behavior in biodegradation of polyethylene in combination with enzymatic hydrolysis of starch. Similar results have been reported previously for biodegradation of starch-PE blend samples in a culturing medium of amylase-producing Vibrios33. The observations of Liu et al. through FTIR-microscope with ATR showed that the carbonyl groups formation mainly appeared around the starch granules in the samples which could be considered as an evidence for the positive role of starch on the polymer degradation12.The weight of samples was measured before and after the degradation process and the obtained results in terms of weight loss percentage have been reported in Table 14. The minimum energy of the system could be achieved when the starch molecules take the V-type conformation which provides an interior hydrophobic space in the center and the hydroxyl groups form an outside hydrophilic surface19  of the PE-starch blend. This could be due to the existence of hydroxyl groups in the starch structure which are hydrophilic while on the contrary PE structure is hydrophobic in natureeim), melting peak temperature (Tpm) and melting extrapolated end temperature (Tefm) which indicates the degree of crystallinity and molecular weight range of the polymer in the samples. The results have been presented in Table Differential scanning calorimetry (DSC) was used to measure the enthalpy of fusion, melting extrapolated onset temperature  was obtained more than that of the untreated samples (A0). Accordingly, more crystallinity has occurred in samples after biodegradation process which is in agreement with the reported results35. In fact, LDPE is characterized by a high degree of short and long chain branching which reduces the crystallinity of the polymer structure. It is believed that branches are more susceptible to degradation which has been supported by the observations in this study as the enthalpy of fusion was higher for the samples after biodegradation. At the same time, melting peak temperature (Tpm) and melting extrapolated end temperature (Tefm) have increased which can be related to chain scission in side groups. In fact, LLDPE is produced by copolymerization of ethylene and higher alpha-olefins such as butene, hexene, or octene. Therefore, the number of carbon atoms in the side chains are more likely to be 2, 4 or 6. It is coming to be generally accepted that increasing the length of the side chains from 1 to 8 can make the molecular packing difficult and the increased flexibility of the side chains causes a reduction in the melting point36. Although, the heat of fusion of samples subjected to the enzymatic degradation was increased in comparison to the untreated samples, the melting extrapolated onset temperature (Teim) has reduced from 94 to 88 °C which in turn, reflects the reduction in the molecular weight of the polymer especially due to the failure in the main backbone of polyethylene chain. Therefore, the biodegradation seems to affect both the main backbone and the side chains of the polymer molecules. In order to have more perception of the effect of alpha-amylase on biodegradation of PE-starch blend, more analysis was carried out through FT-IR spectroscopy.The more the enthalpy of fusion, the higher the degree of crystallinity−1. In polyethylene chain, the stretched C-H absorption occurs just before 3000 cm−1(2840–3000). Methylene and methyl groups have a bending absorption of about 1465 and 1375 cm−1 respectively. At 720 cm−1, the bending motion related to four or more CH2 groups in an open chain appears37. In comparison with untreated samples (A0) as the control, FT-IR spectra displayed a remarkable decrease in OH group in the presence of alpha-amylase (Aα). Moreover, the intensity of peaks at 3000–2840 cm−1 in the treated samples (Aα) decreased and at 1465 and 1375 cm−1, a notable reduction appeared. Further, a significant reduction in 720 cm−1 was observed in Aα, which clarify the weakening of CH2 groups in the polymer chains. The results support our earlier claim that the polyethylene molecules have been degraded in combination with starch hydrolysis in the samples38.The modifications in the chemical structure of PE-starch samples were analyzed by studying their functional groups through FT-IR spectroscopy. The results for the blank and aqueous enzyme solution treated samples have been shown in Fig. 0) and enzymatically degraded samples (Aα) have been calculated by EDS analysis and reported in Table The percentage of carbon and oxygen atoms as well as their weight percent for the control (A6H12O5)n] is 6n/(5n + 1). For the starch with the highest degree of polymerization (n → ∞), this ratio approaches 1.2. The number of carbon atom of polyethylene [(CH2-CH2)m] is 2 m, so the ratio of carbon atoms of polyethylene to oxygen atoms in the starch in the A0 samples (untreated PE-Starch blend) can be calculated about 3.37 according to the following equations:The ratio of carbon to oxygen atoms in the starch by the chemical formula of [ can be determined by equation Similarly, the ratio of carbon to oxygen atoms in polyethylene and starch blend in Aα sample can be estimated between 3.02 to 2.82. Consequently, the decrease of the mentioned ratio from 3.37 to 3.02–2.82 according to the EDXS analysis confirms that polyethylene deterioration has occurred alongside of the starch hydrolysis because the normalized number of the polyethylene carbon atoms has been dropped after the enzymatic treatment.The FT-IR results represent that starch has not been totally hydrolyzed and the ratio of carbon atoms of polyethylene to oxygen atoms of starch in the An, Mp, Mw, Mz, and Mv) of polyethylene for the control (A0) and enzymatically degraded samples (Aα) have been reported in Table 0), all types of the average molecular weights for the enzymatically degraded sample (Aα) have decreased greatly as shown in Fig. −1 were formed in the PE-starch sample after the enzymatic degradation. On the other hand, the weight fraction of molecules having the molar mass less than 4 × 104 has increased while the weight fraction of the heavier molecules has reduced. The dramatic plunge in molecular weight and formation of shorter chain lengths of polyethylene after enzyme treatment indicate the feasibility of the biodegradation of the synthetic polymer which has arisen from the scission event at the polyethylene backbone.The number average, peak average, weight average, z average and viscosity average molecular weights  and enzymatically degraded samples (Aα) were reported in Table 0), a reduction in all types of intrinsic viscosities of the enzymatically degraded sample (Aα) were achieved as demonstrated in Fig. 0) was higher than the enzyme-treated sample (Aα) which indicated that the branching number was smaller for the enzyme-treated sample (Aα). The viscosity difference between the two samples was intensified for heavier fractions of the polymer for upper than 104 g mol−1 molar masses. At the highest molecular weight extreme, the viscosity of the treated sample decreased to one-tenth of the control sample. Similarly, the branching number in the treated sample reduced significantly as can be seen in Fig. 5 and 106, the branching number of the control sample (A0) was higher than the treated polymer (Aα) which demonstrated that the lateral chain branches of polyethylene were affected by the enzymatic degradation so intensive that all of the branches of some polymer chains have been entirely degraded.Moreover, number average, peak average, weight average and z average of the intrinsic viscosity  of polyethylene in the control (A0) and enzymatically degraded samples (Aα) have been reported in Table 0) was seven times higher than the enzyme treated sample in this temperature window which indicated that the highly volatile materials had been removed previously during the enzymatic treatment. In the range of 200 °C to 400 °C, the starch and glycerol content of the blend are mainly affected. The pyrolytic volatilization of the starch occurs in the temperature range of 250 °C to 350 °C and the normal boiling point of glycerol is 290 °C. Therefore, the higher the starch content, the greater the amount of mass changes through thermal gravimetric analysis in the mentioned temperature interval40. Seemingly, the enzyme treated sample (Aα) had a smaller weight loss than the control (A0) because a portion of its starch content had been hydrolyzed by the enzyme. On the other hand, as it was shown in Fig. α) was 32% higher than the control (A0). Evidently, the shorter polymer chains with lower molecular weights and naturally with lower pyrolytic volatile temperatures have been formed in the PE-starch sample after the enzymatic degradation which explains the mentioned observation. At higher temperatures (more than 500 °C), the majority of polymers have been evaporated and both of the samples showed a similar result. Obviously, the obtained TGA profiles in this section are in agreement with the earlier GPC results.Thermal gravimetric analysis (TGA) was employed to analyze the mass changes of the samples as a function of the temperature rise. The percentages of the weight loss in a wide range of temperature variation (from 25 to 800 °C) under a constant heating rate for the control  to the PE structure. However, it has not been clarified that why would a fine powder of PE be still recalcitrant is spite of the large surface area or how can the functional groups be added to the PE molecules while the PE molecules are completely saturated and stable. The obtained results in this work showed that the partial hydrolysis of the V-type starch causes strong hydrophilic hydrophobic repulsions leading to cracks in the polymer structure. Beyond this, it was proved that amylase and starch were effective in degradation of PE molecules. In fact, amylase performs two separate but complementary activities in PE-starch blend degradation. First, the amylase decomposes the starch as its regular specific function and second as a promiscuous function, in the presence of starch breaks the PE molecules. Noticeably, alpha-amylase influenced the degradation of polyethylene indirectly in combination with starch hydrolysis in the blend of PE-Starch while it had no effect on the polyethylene samples without starch. Therefore, the degradation process can be considered as a cometabolic procedure where a complex of starch-amylase or a product of their reaction influences the PE breakdown. However, the exact mechanism has not been known yet and should be studied in detail in the future.Biodegradation of LDPE-starch blend samples by an alpha-amylase aqueous solution were studied. Tensile strengths of the samples after degradation process decreased significantly which was explained by the specific inclusive structure of the blend and the formation of tiny cracks in the matrix of samples because of the strong hydrophobic-hydrophilic interactions between the polymer and starch molecules after partial degradation of the starch. Moreover, the obtained results confirmed the breakdown of PE molecules at the presence of starch and amylase. The presented evidence showed that alpha-amylase in the presence of starch has a non-specific effect on polyethylene molecules. In other words, amylase has a cometabolic behavior in which it hydrolyzes the starch as the primary specific substrate and at the same time, its complex with the starch or a product of starch hydrolysis reaction decomposes the PE molecules. The observed influence of amylase on a non-specific polymeric substrate such as PE has been reported for the first time in this work.2HPO4 and NaH2PO4 were purchased from Merck. The nutrient broth was from Thermo scientific, and unmodified wheat starch was obtained from domestic suppliers (Gol Yas Mashhad-Iran).Alpha-Amylase Liquozyme SC DS was Novozymes production. Glycerol was supplied by Guangzhou Wanjingfeng Company (S. Korea). LLDPE LL0209AA was from Bandar Imam Petrochemical Company (Iran). Na31.Starch is a natural polymer consist of amylose and amylopectin units. Amylose linear structure composes of crystalline and amorphous regions, and amylopectin can be added as branches on the linear part. In order to obtain a thermoplastic product with homogeneous properties, the amylose crystalline structured units can increase by the addition of glycerol as a plasticizer. Moreover, glycerol improves the processability and flexibility of the starch- polyethylene blend. Glycerol increases swelling in the PE-starch blend, thereby improving the water absorption which facilitates the enzyme diffusivity and therefore enhances the susceptibility of the blend3) dimensions were provided for physical testing.Two different series of PE blends (PE-Starch-Glycerol and PE-Glycerol at a weight ratio of 2/1/1 and 3/1) were prepared in a two-step procedure. Firstly, the components were weighed out carefully and added together in a mixer . Then, sheets (thickness of 2 ± 0.1 mm) were produced from the blend using an injection machine under pressure of 20 MPa . The sheets were cut in a dumbbell-shape form according to ASTM D638-10. In addition, rectangular samples with 2 × 8 × 35 . It was added to distilled water in a 1/100 (v/v) ratio to produce the enzyme solution. Three dumbbell-shaped PE-starch blend samples were inserted in the enzyme solution and incubated for 31 days at the ambient temperature while the blank samples were put in the distilled water under the same conditions. After this time, degradation of the samples was analyzed by the relevant physical, mechanical, thermal and chromatography methods.The enzymatic degradation tests were performed in triplicates at 25 °C and neutral pH (7.0 ± 0.5) using an alpha-amylase aqueous solution. The enzyme (Alpha-Amylase Liquozyme SC DS) activity was determined according to Fuwa method0). At the end of the tests period, the samples were washed with distilled water and dried again at 40 °C for 24 h. Then, the final weight of the degraded samples (wd) was measured. The weight loss percentage of specimens after biodegradation was calculated by the following equation:The rectangular samples were oven-dried at 40 °C for 24 h before biodegradation test, and their initial weight was obtained ) in a nitrogen atmosphere as the purge gas according to ASTM D3418-12. 8 mg of the sample was sealed in aluminum pans and subjected to heating condition from 40 to 200 °C at a rate of 10 °C min−1. Three samples for each biodegradation test were analyzed. The DSC analysis provided the enthalpy of fusion and melting point of the polymer samples.The tensile strength was measured according to ASTM D882-12 using a tensile testing machine  at 25 °C. The test speed was 5 mm min−1 and scanning range from 400 to 4000 cm−1 in 25 °C according to ASTM D5477-11.The functional groups of samples before and after degradation experiments were studied through FT-IR spectroscopy. The analytical test was run using FT-IR 6300 apparatus made by JASCO, Germany. FT-IR spectroscopy was carried out using spectral resolution of 1 cm scanning electron microscope (SEM) operating at 15 kV using the secondary electron detector (SE). Quantitative analysis of samples by energy dispersive spectroscopy (EDS) was also performed according to ASTM E1508-12.In addition, microscopic studies of samples were carried out based on the procedure of ASTM E986-04 with a TESCAN MIRA3 XMU VP-FESEM −1. The PE samples were dissolved to obtain concentrations of 1 mg mL−1. The calibration was also performed with Easivial PS-H  which their molecular weights were covering the whole range of 162 and 6000000 g mol−1.Moreover, the polyethylene molecular weight and its distribution were analyzed by GPC  instrument according to ASTM D6474-12 to evaluate the degree of biodegradation. All measurements were recorded using a High-Temperature GPC (HTGPC) system equipped with differential refractive index (DRI), light scattering (LS) and viscometer (VS) detectors. GPC Analysis was performed using 3 PLgel 10 μm Mixed-B (300 × 7.5 mm) columns at 160 °C while 1,2,4-trichlorobenzene  used as the solvent and the mobile phase at a flow rate of 1 mL min−1 and 10 °C min−1 respectively, and the range of the applied temperature was 25 to 800 °C.The final test for confirmation of the irregular function of amylase in biodegradation of the PE-starch blend was thermogravimetric analysis that was accomplished by TGA instrument  as stated in ASTM E1131-08. Nitrogen with purity of 99.9% was used as an inert compressed gas and reactive compressed gas was air. The flow rate and the ramp were adjusted to 90 mL minSupplementary Materials


The fatigue crack initiation position of cast magnesium alloys depends strongly on the radius of curvature of through hole or stress concentration factor at the closed edges of two through holes. In addition, the fatigue multi-cracks were amalgamated for the samples with titled 45° of two small holes of cast Mg-Al alloys when the hole distance is less than 4D .The low cycle fatigue tests on the crack initiation and propagation of cast magnesium alloys with two small holes were carried out by using in-situ scanning electron microscope (SEM) observation technology. The fatigue crack propagation behaviors and fatigue life, which are affected by two small artificial through holes, including the distances between two holes and their locations, were discussed in detail based on the experimental results and the finite element analysis (FEA). The results indicated that the fatigue multi-cracks occurred chiefly at the edges of two holes and the main crack propagation was along the weak dendrite boundary with the plastic deformation vestiges on the surface of α-Mg phase of cast AM50 and AM60B alloys. The fatigue cracking characteristics of cast AZ91 alloy depended mainly on the brittle properties of β-Mg That is to say, the LCF fracture of cast Mg-Al alloys is an intergranular fracture at room temperature  and the micro hardness. The difference in the mechanical properties both α-Mg and β-Mg17Al12 caused the mismatch of deformation and the fatigue cracks occurred easily at the interface [In order to understand the above mention experimental phenomena and characteristics of LCF crack initiation positions and propagation paths of cast Mg-Al alloys (AM60B and AZ91) based on the fatigue damage or fatigue fracture theory, the finite element method (FEM) was carried out as a typical case in nterface ,17,18,19For cast AZ91 alloy, the fatigue crack propagation behavior is more sensitive to changes in the distance between the two small holes than that of cast AM50 and AM60B alloys. This is because the plastic deformation capability of the former is much lower than that of the latter so that the LCF crack propagation resistance of the former is lower than that of the latter. The low cycle fatigue crack propagation behavior of the former depends strongly on the plastic strain or the maximum stress value but that of the latter depends mainly on the von Mises stress amplitude and its distribution.The LCF crack initiation behavior of cast AM60B and AZ91 alloys with tilted 90° and the hole distances indicated that all fatigue crack initiation seats occurred at the edge of hole and LCF crack propagation behavior depends mainly on the distance of two holes, which the LCF crack propagates preferentially between two small through holes when the hole distance is less than λ = 2 mm (4D). Therefore, the correlative effect of the distance of about λ = 2 mm (4D) on the LCF crack propagation can be defined as the critical distance between two small holes when the diameter of hole is about 0.5 mm.max/σ0.2).The LCF crack initiation behavior of cast Mg-Al alloys with tilted angle α = 45° and different hole distances is similar to that of the case with tilted angle α = 90° but the crack propagation behavior depends not only on the distance of two through holes but also on the plastic deformation capability of cast AM60B and AZ91 alloys as well as the applied stress ratio λ parameter  than that of the latter. The results demonstrate a good correlation with S-N curves by using the longitudinal coordinates of the maximum stress amplitude or the λ parameter.The LCF crack growth rates of the cast AZ91 and AM60B alloys are about 1.38 × 10The tests on the fatigue crack initiation and propagation of cast Mg-Al alloys with the two small holes were carried out by using in-situ SEM observation. The experimental results indicate that there are the critical geometric parameters of two small holes to influence on the fatigue behavior of cast Mg-Al alloys and agree with the finite element analysis. Main conclusions are obtained as follows:


An innovative methodology based on non-destructive observation by using harmonic generation microscopy is proposed for detection and location of starch granules and oil in a fried starchy matrix and topography analysis of food products. Specific fluorescent probes were used to label the main biochemical components of the starchy fried matrix, namely starch and oil. Fluorescence of starch and oil respectively stained with Safranin O and Nile red was observed from non-linear microscopy. By using sequential scanning and specific emission filters, it was possible to merge fluorescence and harmonic generation signals. Second harmonic generation (SHG) generated by starch granules was superposed with safranin fluorescence, whereas third harmonic generation (THG), not restricted to the superposition with Nile red fluorescent signal, was used to investigate the topography of the fried product. By these experiments, starch granule mapping and topography of the starchy fried product were obtained without any destructive preparation of the sample. This label-free approach using harmonic generation microscopy is a very promising methodology for microstructure investigation of a large panel of starchy food products.  The distribution of constituents in the matrix of fried foods and the topography of the product are important information to understand phenomena occurring during the frying process. Penetration of oil in the matrix and distribution of non-gelatinized starch are two phenomena usually analyzed in fried products. Bouchon and Pyle  highlighAccording to Pedreschi , oil absHowever, Thanatuksorn et al.  did not Development of surface structure and roughness of fried products is mostly due to the crust formation that creates an increase of the pressure inside the product. This pressure leads to expansion of pores and could generate damage to the surface of the product .Topography analysis could be performed by using scanning electron microscopy (SEM). Lisinska and Golubowska  used imaOil impregnation is usually analyzed by a microscopic observation. In the majority of studies, a dyed oil bath with a thermostable fluorescent probe is used in order to label absorbed oil in the product during the frying process ,9,10,11.Starch gelatinization evaluation is usually made by light microscopy or differential scanning calorimetry (DSC) for observation or quantification of the starch gelatinization respectively . Light mRecently, second harmonic generation (SHG) and third harmonic generation (THG) obtained from nonlinear microscopy have been described as specific signals to identify label-free components in many biological samples ,23,24,25THG was also explored for mapping optical heterogeneities in biological samples ,30. THG The objective of our study was to introduce nonlinear microscopy as a new methodology for structure analysis of a label-free potato fried starchy matrix by investigating the auto fluorescence, SHG from starch granule and THG from interfaces heterogeneities (lipid and surface).The matrix used in this study was prepared from a mix of dehydrated potato and native starch. Dough was formed by mixing these dry ingredients with water. Dough was sheeted using a rolling mill and pieces were cut in the sheeted dough. Pieces of dough were fried at 170 °C in a bath of sunflower oil.Specific staining of starch was performed to validate the harmonic signals attribution to starch granules (SHG). Safranin-O  was prepared at 1 mg/mL in water to stain the starch granules. Safranin-O is not specific to starch but it does not show fluorescence in the absence of starch, so, it has become a starch marker . We addeOil staining was performed by using Nile red  at 1mg/mL in acetone to stain (i) the lipid droplets obtained from sunflower oil/water emulsion (continuous phase water) (ii) the lipid phase of the fried product. We added 0.05 mL of Nile red solution on pieces of 0.5 cm × 0.5 cm of starchy fried matrix. Fluorescence emission wavelength of Nile red is reported in Measurements were performed on a Nikon microscope A1RMP-HD coupled with an Insight Deepsee laser (Spectra Physics), used in the 820−1300 nm range <120 fs pulse width . An auxiliary beam at 1040 nm was available in combination with the tunable output for dual wavelength excitation. For all images, an apochromat 25X MP1300 immersion objective was employed  and distilled water was used as the immersion medium. The microscope was equipped with four non-descanned detectors installed in reflexion mode, three of them were highly sensitive GaAsPs . THG and SHG were performed in epi directions. Different dichroic and emission filters from Semrock were used according to excitation wavelengths and analyzed signals  .For all images presented, we used color assignments as close as possible to true colors for all detection channels. For instance, SHG from native starch granule, observed in blue channel (410/15), occurred from non-linear light matter interaction at 820 nm b and at Starch granules were quantified from SHG and Safranin detection by image analysis with Fiji software . 2D projFluorescence staining of native starch granules was firstly analyzed by multiphoton microscopy from Safranin-O labelling. Safranin-O fluorescence was observed from 1040 nm excitation. The two starch granule populations characteristic of starch were visible with large and small green granules a. SHG fr2 = 0.9877) with an error of 10.8% for the SHG starch granule detection compared to Safranin staining method. On the other hand, in This result was concordant with our acquisition mode for SHG detection which consist in using linearly polarized light with epi-SHG detection limited to only one angle of polarization. Analysis performed on images from fluorescence/SHG detection of starch granule demonstrated that the number of starch granules detected from SHG and Safranin was highly correlated (R2 and 0.11 mm in depth (see Fried starchy product was then observed at 820/1040 nm. Auto fluorescence of the matrix, in this case, the cell wall of a potato, was observed in the yellow channel from 1040 nm excitation a. SHG frepth see c. A supeLipid droplets were firstly stained in an emulsion by using Nile red staining. Nile red fluorescence was observed from 1040 nm excitation a. THG wa2 and 0.36 mm in depth (Imaging of oil in the starchy fried matrix was then performed by using Nile red staining and multiphoton microscopy on an area of 0.3 mmin depth a, The THin depth c.2 and 0.22 mm in depth. (The multiphoton analysis of a label-free fried starchy matrix were performed with dual wavelength excitation, respectively 1240 and 1040 nm on an area of 0.3 mmn depth. . THG wasn depth. a–d.The resulting 3D image showed an irregular surface of the sample in accordance with the macroscopic aspect of the product. This access to the irregular surface structure of the product was possible thanks to the virtual sectioning in depth facilitated by infrared light. As observed on the oil analysis in the matrix, THG gave information about the surface topography because of the air/matrix interfaces. Therefore, a 3D representation of the THG investigation allowed the visualization protrusions and depressions in the sample e. On topA label-free starchy fried matrix was observed thanks to multiphoton microscopy with imaging of harmonic generation signals and endogenous auto-fluorescence generated by the components of the sample. Auto-fluorescence of the starchy fried matrix was due to the phenolic compounds of the potato cell contained in dehydrated potato ,36,37. STHG obtained thanks to the interface’s heterogeneities of the product gave very interesting data on its surface topography with a 3D approach limited to the surface. Surface structure of the fried food product is usually analyzed by SEM ,5,40,41,Finally, from this study, we have demonstrated that SHG and THG could be combined with endogenous fluorescence and fluorescence from specific labeling as oil staining by Nile red fluorescent probe to characterize microstructure of a starchy fried matrix. This approach could provide complementary information on both porosity and its relationship with oil absorption by using oil staining during the frying process which is routinely analyzed to predict the texture of the product ,42.Harmonic generation microscopy shows many advantages compared to other technics, it is a quick and non-destructive method to image intact starch granules after a frying process in the starchy fried matrix and the topography of the surface without any previous sample preparation. This innovative label-free approach could be used to follow the evolution of starch gelatinization process related to the loss of SHG during the transformation of starchy food products and to analyze the surface irregularities of a food product due to THG investigation determinant on food microstructure. SHG detection by using linearly polarized light is limited to about only half of the starch area but we have demonstrated the potentialities of this method from acquisition of Zstack and analysis of maximal projection in 2D. Circularly polarized light would be obviously a best way to investigate entire granule. On the other hand, we demonstrated that THG was not a tool to image specifically oil in label-free fried starchy matrix but could be combine with oil staining for investigation of fat distribution.


We evaluated our results of lengthening of free vascularized fibular grafts using a unilateral external fixator in patients with residual leg length discrepancy after free vascularized fibular graft for lower limb reconstruction.Two patients were administrated to our hospital with residual tibial length discrepancy after vascularized free fibular graft surgery. Lengthening of the free vascularized fibular graft with a unilateral external fixator was performed to correct the leg length discrepancy. Both patients recovered well with no difficult in activities of daily living at the last follow-up.This study shows that lengthening of free vascularized fibular grafts with an external fixator is an effective treatment for massive residual leg shortening after vascularized free fibular graft surgery. Massive segmental bone defects often arise from tumor resection, congenital malformation, trauma, osteomyelitis, and so on –3. ThougA 16-year-old female patient was first administrated to our hospital because her right leg was 18 cm shorter than contralateral side Fig. , Table 1A 17-year-old female patient was administrated to our hospital because her left leg was 9 cm shorter than contralateral side Fig. . When shThe surgical management of large bone defect is challenging for orthopedic surgeons. Several different treatment options for this complex disease, such as autografts, allografts, induced membranes, microvascular osseous transfer, as well as bone transport , 22–24. Though FVFG is a reliable method for bone defect, it is also associated with a high complication rate Table . The mosThe mechanical performance of vascularized fibular transfer-based lower-limb reconstruction is dependent on the ability of the graft to hypertrophy. FVFG hypertrophy has been well studied. El-Gammal et al. conducted a study on 25 patients with lower limb tumors who underwent reconstruction with vascularized fibula graft, and suggested that hypertrophy of the vascularized fibular graft is a time-related phenomenon and generally affected by the age of the patient . HoweverBased on the criteria recommended by Paley et al. , 21, theThe optimal time to perform lengthening on a transferred bone is also a main issue that should be taken into consideration by the orthopedic surgeon Table . De BoerThere are no guidelines for the lengthening of a FVFG. We adopted the same lengthening protocol as that for native bone . In ordeIn conclusion, our study shows that lengthening of FVFG with an external fixator is an effective treatment for massive residual leg shortening after vascularized free fibular graft for lower limb reconstruction.


This study aimed at comparing the mechanical properties of conventional and locking dual plates in adjacent and orthogonal orientations for the surgical fixation of transverse femoral shaft fractures. It also assessed the failure mechanics after dual adjacent and orthogonal locking plate removal. t-test and one-way analysis of variance for the comparison of means between two or more groups, respectively. Thirty-two composite femurs were transversally osteotomized and randomly assigned for fixation with either dual locking or compression plates in an adjacent or orthogonal configuration. Sixteen specimens were preloaded axially to 20 N and single-leg stance loads were simulated. The remaining sixteen constructs were subjected to torsional loads of 10 Nm at a rate of 10 Nm/s in external and internal rotation of the femoral head in relation to the knee. Overall combined rotational stiffness was calculated. Eight different specimens with no osteotomy underwent the same experiments after dual locked plate removal and were tested to failure in combined eccentric axial and torsional modes. Data were statistically processed using a two-tailed Orthogonal constructs were statistically stiffer in axial loading compared to their adjacent counterparts in both conventional and locking configurations (p<0.001). Dual locking plates provided higher torsional stiffness than conventional ones within each plate orientation (p<0.01). Neither axial/torsional strength nor failure loads differed between constructs that had adjacent or orthogonal dual locking plates instrumented and then removed (p>0.05). In both orthogonal and adjacent orientations, double locking plates provide higher stability than their dual conventional counterparts. Orthogonal dual plate configuration is more stable and biomechanically superior to dual adjacent plating for constructs fixed with either standard compression or locking plates. Femoral shaft fractures are common lower-extremity insults that typically result from high-energy injuries . They arCompression and locking plates utilize different mechanisms to attain diaphyseal fracture reduction and healing  . The forSingle-plate osteosynthesis may be ineffective in achieving adequate reduction of fractures subjected to increased loads, a finding that led some authors to recommend dual plating in such circumstances , 10, 15.The objectives of this study were to compare the mechanical properties of conventional and locking dual plates in an adjacent or orthogonal orientation in the surgical reduction and fixation of femoral diaphyseal fractures. This investigation also assessed the strength and failure mechanics of the femur after dual adjacent and orthogonal locking plate removal.This biomechanical project involved neither live animals nor human participants and did not require Institutional Review Board approval prior to its completion.Thirty-two composite third-generation medium left femurs  were randomly assigned for fixation with 1 of 4 distinct plate configurations .A diaphyseal femoral midshaft fracture was simulated by performing a transverse osteotomy. The fracture was spanned and stabilized with either 10-hole 4.5 mm stainless steel dynamic dual compression plates (DCP)  or dual locking plates (DLP) . Axial and torsional analyses included 16 identical specimens for each mode, with 4 sets of 4 composite femurs .Plates were placed 8.5 cm distal to the tip of the greater trochanter along the lateral side of the femur. Pilot holes, 3.5 mm in dimension, were drilled into each of the outermost 4 holes on the proximal and distal sides of each plate. Eight bicortical 4.5 mm screws per plate were then inserted with a torque wrench into each of the pilot holes, securing the plate to the synthetic bone. After instrumentation of the lateral plate, a hacksaw was used to create a 1 cm osteotomy gap in the middiaphysis of the femur and center of each 10-hole plate so that the closest holes to the defect could be left empty as per the surgical protocol , 18. TheTo assess the strength and failure mechanics of the femur after locking plate removal, the second experimental arm consisted first of instrumenting dual locking plates in either an adjacent or an orthogonal orientation (4 synthetic femurs in each group). In both configurations, the plates were centered in a similar fashion as described above, not spanning any diaphyseal femoral midshaft osteotomy. The screws and plates were then removed. A total of 8 synthetic constructs, each containing 16 holes (8 holes per plate) in an adjacent (4 specimens) or orthogonal (4 specimens) orientation, were therefore generated.For axial loading , the condyles and distal ends of each synthetic femur were potted with polymethylmethacrylate (PMMA) in an aluminum box in 10 degrees of adduction relative to the anatomical femoral axis. Femurs were rigidly secured distally to the load cell of a servohydraulic materials testing machine  in a custom loading frame that allowed force transfer along the biomechanical axis of the femur . Axial lFor torsional loading , the composite femurs were potted distally as before, while the head and greater trochanter were set within a custom aluminum plate channel that would allow rotational forces to be applied proximally. Each construct was loaded to 10 Nm at a rate of 10 Nm/s in both external and internal rotation of the head in relation to the knee . Load diAfter being subjected to the biomechanical testing protocol described above to determine axial and torsional stiffness, the eight 16-hole constructs with no plates were then tested in combined eccentric axial compression and torsion until failure. Peak combined axial and rotational failure loads were recorded and analyzed. t-test. Post hoc analysis was also conducted to assess the power of the study. All analyses were performed using SPSS® version 24 .Data were normally distributed, justifying the use of parametric statistical tests. Comparisons of axial and torsional stiffness between the different plate configurations and types were computed with a one-way analysis of variance (ANOVA) and a post hoc Scheffé test for intergroup analysis. Comparisons of axial/rotational stiffness and failure torque between adjacent and orthogonal constructs with the plates removed were computed with a two-tailed Student'sThe instrumentation and subsequent testing were completed according to the protocol described. All constructs were successfully tested as intended with no outliers. In axial loading, orthogonal plate configurations provided significantly superior compressive stiffness than adjacent orientations in both conventional and locking-plate subgroups. Group 1 (DCP-A) exhibited an axial mean stiffness of 364.9 N/mm (95% CI 311.2–418.5 N/mm), compared to 702.1 N/mm (95% CI 443–961.2 N/mm) in Group 2 (DCP-O) (p<0.001) . SimilarIn torsional testing, DCP-O constructs (Group 2) exhibited higher stiffness compared to their adjacent DCP-A counterparts (Group 1), with mean values of 3.7 Nm/deg (95% CI 3.1–4.2 Nm/deg) and 2.9 Nm/deg (95% CI 2.8–3.1 Nm/deg), respectively (p<0.01) . Group 3Dual locking plate removal demonstrated no statistical differences in axial and torsional stiffness among the resulting constructs. Adjacent holes specimens showed a mean axial stiffness of 1100 N/mm (95% CI 1011.5–1190.1 N/mm) compared to 1136.8 N/mm (95% CI 1075.6–1197.9 N/mm) for constructs with orthogonal holes (p=0.53). Similarly, specimens with adjacent holes exhibited a mean torsional stiffness of 7.7 Nm/deg (95% CI 7.4–8 Nm/deg) compared to 7.4 Nm/deg (95% CI 7–7.7 Nm/deg) for constructs initially instrumented with plates in an orthogonal orientation (p=0.2). Orthogonally configured constructs failed at an average of 5056.2 N (95% CI 4315.1–5797.3 N) axially at a 25 Nm (95% CI 21.1–29 Nm) of torque, whereas their adjacently oriented counterparts broke at an axial force mean of 4764.3 N (95% CI 4249.3–5279.2 N) and a torque of 23.9 Nm (95% CI 21.1–26.5 Nm) .Femoral shaft fractures are of special concern in elderly individuals, who are prone to osteoporosis and other associated comorbidities. These common traumatic injuries are most frequently treated with open internal surgical reduction, which aims at providing a stiff and rigid construct while allowing early patient mobility –21. FixaDual plating is a surgical fixation method that has been mainly used for the management of distal and proximal humeral fractures , 28–31. A retrospective study investigated locked implants in staged dual plating of traumatic open supracondylar femoral fractures and revealed a 100% union rate in all 15 patients analyzed . This fiIn axial compression, our results indicate that dual orthogonal plating constructs were stiffer compared to dual adjacent plating fixations. Orthogonal dual compression constructs exhibited higher stiffness compared to dual adjacent locking constructs, which highlights the relevance of the plate orientation in reducing femoral shaft fractures. In rotational load testing, both locking and compression orthogonal constructs had higher rigidity compared to their respective parallel counterparts, but this difference reached statistical significance in the conventional compression plate subgroup only. Taken together, our data suggest that orthogonal plating yields higher biomechanical stiffness than adjacent constructs, with dual locking orthogonal plating outperforming all other surgical fixation techniques. Our findings are in disagreement with the results of Jazrawi et al., who documented that locking plating had similar fixation stability compared to their dynamic compression counterparts in axial loading . These dDual locked plate removal did not yield any difference in axial and torsion loading between orthogonally and adjacently configured composite femurs. Similarly, peak combined axial and rotational failure loads were comparable between both groups. These interesting findings were opposite to our initial expectation, in which we postulated that the relatively smaller distance between adjacent screw holes might induce a higher local stress raiser that requires lower energy to fail, compared to the longer distance created in orthogonally oriented constructs. The failure risk being associated more with the screw diameter or number, rather than their location, might explain our results.Our study is subject to some limitations. While the sample size of composite femurs may have appeared relatively small, all biomechanical metrics analyzed to address our primary hypothesis were well powered. A post hoc analysis revealed 98% to 100% power in almost all measures. The only underpowered evaluation (74%) was the torsional load stiffness comparison between orthogonal and adjacent dual locking plates. It is possible that we might have been able to identify a statistically significant difference between these construct groups with a larger sample. Moreover, composite femurs may not reflect the bone biology of the osteopenic elderly patient population, and the testing methodology did not simulate all musculotendinous forces on the femur that would be found in vivo. The second experimental arm aimed at analyzing fractured femurs that have completely healed. This might be subjected to some drawbacks, in that the fracture site might still be fragile and the bone quality not totally restored. However, this design allowed us to study a homogenous biomechanically human-equivalent and validated synthetic femoral model without the need to account for interspecimen volumetric variations or assess the bone mineral density, a confounding variable commonly encountered with human cadaveric studies . AdditioIn summary, orthogonal and adjacent dual plating are appropriate operative techniques for femoral shaft fracture fixation. Locking plates allow higher construct rigidity and stability compared to conventional dynamic compression plates. Clinical studies with comparison of both surgical modalities are needed to analyze the potential complications' incidence and functional outcomes.


However, the effect of N fertilizer on the physicochemical properties of starches isolated from this cultivar has not been reported. In order to evaluate these effects, three different N rates, 0 , 75 (N1), and 150 kg ha-1 (N2), were selected for a field experiment in 2017. The results showed that N1 exhibited the highest storage root yield and starch yield. Compared to the control group, N fertilizer significantly increased the total starch content while no significant difference was found in these between the N1 and N2 groups. The amylose (AM) content was highest in the N2 group and lowest in the N0 group. In addition, N fertilizer exhibited no significant effects on the values of [D], D  and D . Compared to the control group, N1 demonstrated significantly higher setback viscosity (SV), while N2 showed significantly higher peak viscosity (PV), cold paste viscosity (CPV) and SV. However, there were no significant differences in the hot paste viscosity (HPV), peak time and pasting temperature between the N1 and N2 groups. For the thermal properties of starch, there were no significant differences in peak temperature (Tp), conclusion temperature (Tc) or gelatinization enthalpy (ΔH) between the N1 and N2 groups. Overall, for the starch samples of cultivar Jishu 25, N fertilizer exerts significant effects on the starch content, AM content and viscosity properties but little effect on the particle size distribution and ΔH. 75 kg N ha-1 can easily lead to substantial planting benefits from the high storage root yield, dry matter yield and total starch content of this cultivar.In recent years, the sweet potato cultivar Jishu 25 has exhibited good characteristics for starch processing in northern China. The storage root dry matter yields of this cultivar can exceed one ton per mu (1/15 of a hectare) at nitrogen (N) rates of 60–90 kg ha Sweet potato has been used globally for vegetable and staple food production and as a raw material for industrial processing . Sweet pIn China, the sweet potato harvest area is 3281.52 thousand hectares, and the total production was 70.57 million tons in 2016, ranking first in the world . The pro-1 was required for maximum root yield of 'Beauregard' sweet potato in Virginia. In subsistence sweet potato production systems of humid tropical Papua New Guinea, mineral N fertilizer 25 kg N ha-1 plus poultry manure 25 kg N ha-1 produced significantly greater total tuber yields than in controls , 50th, median [D] and 90th [D] percentiles, as well as surface weighted mean diameter D and volume weighted mean diameter D  , 23.o), peak temperature (Tp), conclusion temperature (Tc) and gelatinization enthalpy (ΔH) were determined  values were not significantly different among the three groups. Likewise, D  and D  values exhibited similar trends as those of [D]. These results indicate that N fertilizer exerts minor effects on the particle size distribution of starch granules.The starch granules of cultivar Jishu 25 displayed a unimodal size distribution and ranged from 5 to 60 μm , with mo-1 increased the SV level, while 150 kg N ha-1 elevated the levels of PV, HPV, CPV and SV and reduced the pasting temperature. The two N rates of 75 and 150 kg ha-1 had no significant effects on HPV, peak time or pasting temperature.The pasting profiles of starches were determined using RVA , and theo, Tp, Tc and ΔH, are presented in o value was highest in the N0 group and lowest in the N2 group. The Tp value in the N0 group was significantly increased compared to in the N2 group, and no significant difference was found between the N1 and N2 groups. Tc values in the N0 and N1 groups were significantly higher than in the N2 group, and there was no significant difference in the Tc value between the N0 and N1 groups. Additionally, ΔH values were not significantly different among the three N rates. These results suggest that N fertilizer decreases To values, and 150 kg N ha-1 further reduces the values of To and Tc. However, N fertilizer exhibits no significant effect on ΔH.The DSC curves under different N rates are shown in -1 [-1 may not greatly decrease the contents of starch and AM but a rate of 180 kg N ha-1 significantly reduces the storage root yields of both cultivars [-1, respectively, under N application rates of 0–150 kg ha-1. A application rate of 75 kg N ha-1 produced significantly higher total starch content and lower AM content than those without N cultivation. Raising the N rate to 150 kg ha-1, produced no significant difference in total starch content but starch yield was significantly decreased because of the reduced storage root yield. Additionally, the highest dry matter accumulation in tuberous roots cultivated with 75 kg N ha-1 was 1049.79 kg per mu, which exceeded one ton per mu. Therefore, applying 75 kg N ha-1 easily achieves high planting benefits, as revealed by the high yields of storage roots, dry matter and total starch for cultivar Jishu 25.Starch yield is a parameter for determining the benefit of planting sweet potato. The use of high-starch cultivars by farmers needs to be encouraged by the processing industries, and farmers should be reawarded with appropriate performance incentives . For swe-1 . However-1 exhibited no significant effects on the parameters of particle size, while 150 kg N ha-1 decreased both the 10th and 50th percentiles when compared to the particle size in plants without N application. The two rates of added N fertilizer revealed no significant difference in these parameters, suggesting that different N rates have little effect on the particle size distribution of cultivar Jishu 25.Hoover  produced-1 significantly increased the levels of PV and CPV. Compared to 60 kg N ha-1, the excessive N rate of 240 kg ha-1 significantly increased the levels of PV, HPV and CPV for cultivar Xushu 22 [-1 increased the SV level compared to non-N cultivation, and elevating the N rate to 150 kg ha-1 further increased the levels of PV, CPV and SV. This maybe related to the lower AM content of the starch samples caused by applying 150 kg N ha-1. Previous studies have shown that the ΔH of sweet potato starches ranges from 7.6 to 15.8 J/g [ΔH [ΔH of Jishu 25 starch ranged from 9.81 to 10.30 J/g, which falls within the defined range for this species. N fertilizer significantly decreased the AM content of the starch samples and affected some of the parameters of pasting property. However, 75 or 150 kg N ha-1 did not significantly affect ΔH values. This suggests that the ΔH is affected by many aspects of starch granule gelatinization and it is difficult to link ΔH to only one factor. Moreover, the N rate of 150 kg ha-1 with a lower transition temperature exhibited no significant changes in ΔH compared to the control with a higher transition temperature, which is incompatible with the results of Abegunde et al. [The pasting properties, such as PV, BV, SV and peak time varies significantly among sweet potato starches . LikewisXushu 22 . In this15.8 J/g , 27. Fur J/g [ΔH . In thise et al. . This in-1) with low AM content exhibited higher PV, CPV and SV levels and lower pasting temperature compared to those in the control group with high AM content. These results were consistent with data from only some previous studies [-1 is the most cost-effective N application rate.Starches with different AM contents are of great interest due to their wide applicability and impacts on final product characteristics . Starche studies , 32. Gen studies . Our res studies –7. Addit studies . In thisΔH and particle size distributions  of the starch samples. Notably, 75 or 150 kg N ha-1 had no significant difference on the mean diameter of starch particles, HPV, peak time, peak temperature and ΔH of the starch samples. Additionally, the 75 kg N ha-1 treatment resulted in the highest dry matter content in the storage roots and yielded the greatest amounts of starch and storage roots, indicating that it is a cost-effective N rate for cultivating Jishu 25.Overall, N fertilizer exerts significant effects on the total starch content, AM content and viscosity properties  of the cultivar Jishu 25 but little effect on the S1 File(XLS)Click here for additional data file.S2 File(RAR)Click here for additional data file.


Alcohol Health & Research World introduces both a new title and a new look. These changes reflect a series of improvements that have occurred in the journal’s format and content over the past several years that are designed to maintain and improve the journal’s relevance and utility to our readers. Since its inception in the 1970s, the journal has evolved from a general interest magazine to an internationally known peer-review science journal. This evolution reflects the changing mission of the National Institute on Alcohol Abuse and Alcoholism (NIAAA) from a general purpose alcohol agency to an internationally respected research Institute. It also reflects the changing nature of the alcohol field and the growing need for science-based information to better understand and improve our ability to prevent, treat, and reduce the problems associated with alcohol abuse and alcoholism.This issue of Alcohol Research & Health.In recent years, we have taken great care to assure that the material presented in the journal is informative as well as scientifically sound. This challenge has included presenting technical findings in ways that are interesting and useful to the wide range of disciplines and agendas that comprise our audience, including educators, counselors, clinicians, researchers, and policymakers. As we have progressed to a fully refereed journal, we also have become aware that the title of the journal does not truly reflect the full scope and substance of the information presented in each issue. With this in mind, we recently invited readers and members of the alcohol research field to submit ideas for a new title commensurate with the evolving “ flavor” of the journal. With this issue, we are pleased to announce that a new title has been selected. Please be welcomed to Though not very different from our original name, we feel that this new title will give readers a more accurate sense of our journal’s mission. We are about research—that is, the information we feature is evidence based and fully reviewed by professionals in the field. We also are about health in that our ultimate goal is to disseminate the science that informs the practice of caring for and about people affected by alcohol problems.In launching the new title, we also have instituted some minor design changes. These modifications are intended to make the material presented in the journal more “reader friendly.” We hope you like the new title and the corresponding new design. We look forward to continuing to provide the most up-to-date research findings on topics that are important in alcohol research.


Background. Effects of veneering porcelain thickness and background shade on the shade match of zirconia-based restorations are unclear. The aim of this study was to evaluate the impacts of veneering porcelain thickness and background shade on the shade match of zirconia-based restorations.Methods. Forty A2 shade veneered zirconia disk specimens (10 mm in diameter) were fabricated, with veneering porcelain thicknesses of 1.6, 1.8, 2.0 and 2.2 mm. Three backgrounds were made of A2 shade composite resin (A2), nickel-chromium alloy (NC) and amalgam (AM). The veneered zirconia specimens were placed on the backgrounds. CIELab values were measured with a spectrophotometer. ΔE values were measured to determine color differences between the specimens and the A2 VITA classical shade (target shade). ΔE values were compared with an acceptability threshold (ΔE=3.7). Repeated measures ANOVA, Bonferroni, and 1-sample t-test were used to analyze data (P<0.05).Results. Mean ΔE values ranged between 1.9 and 5.0. The veneering porcelain thickness, the background shade and their interaction affected the ΔE (P<0.0001). The minimum veneering porcelain thickness for the shade match was 2 mm for NC and 1.8 mm for AM.Conclusion. Veneering porcelain thickness and background shade affected the shade match of zirconia-based restorations. With dark-shaded backgrounds, the amount of veneering porcelain thickness needed for the shade match might be beyond acceptable clinical limits. Tooth-shaded backgrounds are esthetically advocated rather than dark-shaded backgrounds in zirconia-based restorations. Zirconia copings represent benefits of high fracture strength,4 proper optical properties5 and a white-to-ivory color.6 Veneering porcelains create a tooth-like appearance due to their proper shade, translucency and tooth-like appearance.7 Consequently, zirconia-based restorations have become popular in dentistry.8Zirconia-based restorations are an acceptable treatment option in restorative dentistry because of their appropriate strength and esthetics.*, a* and b* denote lightness, redness-greenness, and yellowness-blueness, respectively.10 Additionally, the CIELab is used to determine the color difference between two objects. A single value, known as ∆E, is calculated from the formula: ΔE*ab= [(L*2- L*1)2+ (a*2- a *1)2+ (b*2- b*1)2]1/2 in order to measure the color difference.10 Thereafter, the ∆E is compared with acceptability and perceptibility thresholds in order to evaluate the visibility of the color difference to human eyes.14 The CIELab system has been reasonably employed to determine the shade reproduction of different ceramics and restorations.18In order to quantify the color of an object, different color systems have been developed in color science. One of the most commonly applied color systems in dentistry is CIELab, in which L10 Depending on the shade, brand and thickness, zirconia ceramics exhibit different absolute translucencies (visible light transmittance percentage) from low (20%) to ultra (49%) levels. 20 Therefore, zirconia might manifest the color of its underlying materials such as backgrounds and luting agents, leading to an improper shade for zirconia-based restorations.9 A 1-mm-thick zirconia ceramic is needed to create an acceptable masking ability regardless of background color.21 The background L* value affects the masking ability of an 0.5-mm-thick white zirconia ceramic.22 A zirconia-based restoration commonly includes a 0.3- to 0.5-mm-thick zirconia coping and a 1-mm-thick veneering porcelain. Although an 0.4-mm-thick zirconia coping alone produces acceptable masking ability on tooth-colored backgrounds, it might be insufficient for masking metal backgrounds.23 Zirconia-based restorations with an 0.4-mm-thick coping might not be color-matched with natural teeth when placed on gold alloy posts and cores.24 An 0.4-mm-thick zirconia coping alone represents a relative masking ability on color backgrounds, which might lead to color mismatches;24 however, when this zirconia coping is layered with an 0.4-mm-thick veneering porcelain, the resultant color might be further adjusted.25 Moreover, the shade of zirconia-based restorations on a dark background might depend on veneering thickness.26 Some color mismatches have been reported at the cervical area of zirconia-based restorations with 0.3 and 0.5-mm-thick copings.27 Controversially, no significant differences have been reported between CIELab values of zirconia-based crowns on metal backgrounds and composite resin backgrounds.28Zirconia ceramics are optically semi-translucent materials.Various backgrounds and veneering porcelain thicknesses might be used in zirconia-based restorations, inducing different color results. However, according to the controversial results derived from the literature, effects of veneering porcelain thickness and background shade on the shade match of zirconia-based restorations are not clearly understood. Therefore, the aim of this in vitro study was to evaluate the effects of veneering porcelain thickness and background shade on the shade match of zirconia-based restorations. The null hypothesis was that the veneering porcelain thickness and background shade would not affect the shade match of zirconia-based restorations.23 Thus, 40 veneered zirconia disk specimens with 0.4 mm zirconia thickness and 4 veneering porcelain thicknesses  were fabricated. The range of veneering porcelain thickness was selected based on a pilot study.A sample size of n=10 was determined in each study group by considering α=0.05, β=0.1, the study design and previous studies.A computer-aided design/computer-aided manufacturing system  was employed to mill zirconia blanks  and to prepare zirconia disks. The zirconia disks were 0.4 mm in thickness and 10 mm in diameter. The zirconia disks were shaded with an A2 shade coloring liquid  through a 2-minute-immersion process. The zirconia disks were sintered at 1480°C through a 12-hour process in a sintering furnace  and were then adjusted to achieve the intended thickness of 0.4±0.02 mm using a zirconia polishing kit . The zirconia disk was eliminated from the study in case of lack of the intended thickness. Finally, the zirconia disks were cleaned in a 98% ethanol solution and air-dried.An A2 shade feldspathic veneering ceramic for zirconia frameworks  was used to veneer zirconia disks with regard to intended thicknesses using manual add-on technique. The veneered zirconia disks were fired for 69 minutes at a heat rate of 55°C/min from 500°C to 910°C, cooled to room temperature, and polished by using a porcelain polishing laboratory kit . A 3-step polishing procedure was performed using green, purple and yellow polishing disks, as instructed by the manufacturer. The veneered zirconia disks were adjusted to achieve the intended thicknesses (±0.02) using the same porcelain polishing/adjusting kit. The veneered zirconia disk was eliminated from the study in case of lack of the intended thickness. The veneered specimens were cleaned and dried with the aforementioned methods.2 to fabricate the composite resin background (A2). A wax pattern was cast to prepare the nickel‒chromium background (NC). The amalgam alloy was triturated and condensed in a plastic mold to fabricate the amalgam background (AM). All the backgrounds were 10 mm in diameter and 10 mm in height  .Three cylindrical backgrounds were made from an A2 shade light-polymerized composite resin , a nickel‒chromium alloy , an amalgam alloy  Yellow Caps, Densply Sirona, York, PA, USA). The composite resin was applied to a plastic mold and incrementally polymerized with a light-polymerizing unit  for 40 seconds with an intensity of 800 mW/cmn height .21,22 CI23 This mold prevented external lights, supplied a seat for the backgrounds and matched the conditions of spectrophotometry for all the specimens.24 The specimens were seated on the backgrounds without an intermediate.25 Then the CIELab color measurements were performed by an expert operator at the center of the specimens 3 times for each specimen and the average values were recorded .30 The spectrophotometer confirmed the A2 shade for the tab. In order to determine the color difference between the specimens and the target shade, ∆E was calculated from this formula: ΔE*ab= [(ΔL*)2+ (Δa*)2+ (Δb*)2]1/2. An acceptability threshold of ΔE=3.711 was assumed to assess the color differences between the specimens and the target shade and to judge the specimens’ shade match.The same spectrophotometer and a customized putty mold were employed for color measurments.recorded . Also thData were analyzed with SPSS 21 . Kolmogorov-Smirnov test indicated the normal distribution of data in all the studied groups (P>0.05). The effects of veneering porcelain thickness, background shade and their interaction on the CIELab and ∆E values were evaluated using repeated-measures ANOVA. Pairwise comparisons of the studied groups were performed using the Bonferroni correction. The ΔE values of the studied groups were compared with the threshold for acceptability (ΔE=3.7) with STATA  using one-sample t-test. The 0.05 level of significance was considered for all the tests.The mean CIELab and ΔE values for the veneering porcelain thicknesses of 1.6, 1.8, 2.0, and 2.2 mm for the studied backgrounds  are presented in One-sample t-test was used to compare the means of ΔE values with the acceptability threshold (ΔE=3.7). The null hypothesis of ΔE≤3.7 was rejected for NC in 1.6 (P<0.0001) and 1.8 (P<0.0001), and for AM in 1.6 (P<0.0001), while it was not rejected for NC in 2.0 (P=1) and 2.2 (P=1), for AM in 1.8 (P=0.744), 2.0 (P=1), and 2.2 (P=1), and for A2 in all thickness groups (P=1).According to the results of this study, which indicated significant differences in the CIELab and ΔE values in relation to veneering porcelain thickness and background shade, the null hypothesis of the study was rejected. With 0.4-mm zirconia coping, the minimum porcelain thickness for the shade match was 2 mm for NC and 1.8 for AM, while all the tested thicknesses resulted in shade match for A2.9 Veneering porcelains are more translucent than zirconia.9 Thus, a background might express its color under the zirconia coping, affecting the resultant color of a zirconia-based ceramic. The resultant color is the outcome of the ceramic and background colors. Since the backgrounds exhibited different CIELab values, they affected the resultant color in different degrees. The background color initiates the difference in the minimum veneering porcelain thickness needed for esthetics. A greater color difference between the background and the target shade needed a greater veneering porcelain thickness to gain a proper shade match. This is why the general ranking for the proper veneering porcelain thickness needed for the backgrounds was NC>AM>A2  on metal backgrounds and composite resin backgrounds. Their result on the background’s effect was consistent with the present study. Both studies showed that increasing the total restoration thickness beyond the clinically recommended restoration thickness (1.5 mm) led to the color match in zirconia-based restorations regardless of the background color.Suputtamongkol et al23 assessed the effect of zirconia coping thickness and background type on the color masking ability of zirconia-based restorations. They reported that the thickness of zirconia coping should be at least 0.8 mm in order to attain an ideal masking ability on nickel-chromium alloy, while it could be 0.4 mm for tooth-colored backgrounds. However, the present study remarked the veneering ceramic thickness for shade matching. Since the translucency of feldspathic ceramics is more than that of zirconia ceramics, greater thickness for feldspathic ceramics than for zirconia ceramics is needed to achieve an ideal masking ability. However, feldspathic ceramics are optically more similar to tooth structures of dentin and enamel than zirconia ceramics.9 Therefore, the shade/translucency matching of zirconia-based restorations seems more achievable with increasing the veneering ceramic thickness than with increasing the zirconia coping thickness. However, both studies confirmed an increase in the restoration thickness with dark-shaded backgrounds for esthetics.Tabatabaian et al24 reported that background shade , total ceramic thickness (1 and 1.5 mm), and zirconia coping brand  affected the resultant color of zirconia-based restorations with 0.4-mm-thick zirconia coping layered with IPS e.max Press Ceram. They showed that only Lava crowns on gold alloy cores could not create a color match, because their ∆E color difference values were more than the acceptability threshold (close to 5.5). The discrepancies in the results of the studies on the effects of ceramic thickness and background shade might be attributed to the difference in zirconia coping and veneering ceramic brands, control groups, and acceptability thresholds used by the studies.Oh and Kim32 Therefore, with consideration of both esthetic and mechanical properties, the use of tooth-shaded backgrounds rather than dark metal backgrounds is recommended for zirconia-based restorations. An increase in zirconia coping thickness might be a solution for the esthetics in zirconia-based restorations, which was not evaluated in this study. The assessment of the effect of zirconia coping thickness on the color match of zirconia-based restorations is suggested for future research studies.According to the results of this study, increasing the veneering porcelain thickness of zirconia-based restorations improves the esthetic outcomes; however, this might increase the risk of porcelain chipping.9 Among these factors, veneering ceramic thickness and background shade were investigated in this study and accordingly, using a background with a shade corresponding to the target shade is advised instead of dark-shaded backgrounds  for zirconia-based restorations. In case of a dark-shaded background, increasing the restoration thickness might be considered. However, an increase in zirconia coping thickness might be more advantageous than an increase in veneering porcelain thickness alone regarding both mechanical and esthetic properties.The color of zirconia-based restorations might be affected by factors such as background, luting agent, zirconia coping , veneering ceramic , glaze, surface staining, and laboratory procedures .This study did not evaluate some affective factors such as luting agent, thickness and brand of zirconia coping, brand of dentin veneering ceramic, enamel veneering ceramic, glaze, veneering technique, and external staining. Therefore, evaluation of these factors is suggested for future investigations.Within the limitations of this in vitro study, the following conclusions were drawn: Veneering porcelain thickness and background shade affected the shade match of zirconia-based restorations.With dark-shaded backgrounds, the amount of veneering porcelain thickness needed for the shade match might be beyond the acceptable clinical limits.In order to create the shade match for zirconia-based restorations, tooth-shaded backgrounds are advocated rather than dark-shaded backgrounds.The authors declare no conflict(s) of interest related to the publication of this work.FT contributed to the concept and design of the work. The acquisition, analysis, and interpretation of data were accomplished by FT, KA, and MN. FT, KA, and MN drafted and revised the manuscript critically for intellectual content. All the authors read and approved the final manuscript.This study was supported by a grant from the Research Deputy of School of Dentistry of Shahid Beheshti University of Medical Sciences, Tehran, Iran [grant number 11325].The authors thank the Research Deputy of School of Dentistry of Shahid Beheshti University of Medical Sciences for the financial support (the grant No. 11325), Mehr Dental Lab for their CAD/CAM support, and Mr Aliakbar Deghost for his laboratory work in preparing specimens.Not applicable.


Background: Interaction between cancer cells with microenvironment is essential for cancer progression, therapeutic resistance and prognosis. Chemokine CXCL1 shows variable roles in the development of cancers. DACH1 has been considered as a tumor suppressor and represses the expressions of several chemokines. The relationship between CXCL1 and DACH1 in non-small cell lung cancer (SCLC) deserves further investigation.Methods: Immunohistochemistry staining was performed on tumor tissue microarrays from lung cancer patients to detect CXCL1 protein. The CXCL1 concentration in the serum of adenocarcinoma patients was measured by ELISA. The CXCL1 protein secreted by cancer cell lines was detected by SearchLight proteome array and human cytokine antibody array. The meta-analysis of CXCL1 expression form public databases was performed and correlation between CXCL1 and DACH1 was analyzed. Moreover, the association between clinicopathological features and prognosis with CXCL1 and DACH1 was analyzed by tissue array and KM-plotter from public database.Results: The protein abundance of CXCL1 in lung cancer tissues was significantly higher than that in adjacent normal tissues. CXCL1 was closely related to TNM stage, tumor size, and lymph node metastasis and predicted worse overall survival in adenocarcinoma. The level of CXCL1 in the peripheral blood of adenocarcinoma patients also significantly elevated and positively related with clinical stage. The meta-analysis demonstrated that CXCL1 mRNA level was increased in lung cancer tissues and high level of CXCL1 indicated tumor progression in lung adenocarcinoma. In addition, public database analyses showed that CXCL1 negatively correlated with DACH1. Stable overexpressing DACH1 in cultured lung cancer cells remarkably decreased CXCL1 protein. Moreover, ectopic expression of DACH1 significantly inhibited the expression of CXCL1, Ki67, and cyclin D1 in tumor tissues compared with A549 cells with empty vector. Survival analysis showed that high CXCL1 and low DACH1 indicated poor overall survival and progression-free survival.Conclusion: CXCL1 is closely associated with tumor progression and poor survival. DACH1 significantly inhibits the expression of CXCL1 and indicates good prognosis. Therefore, combined detection of CXCL1 and DACH1 could more precisely predict prognosis of lung adenocarcinoma. Lung cancer is the most common cause of cancer death in male and the second leading cause in female all over the word . Lung caHuman Dachshund homolog 1 (DACH1) is a key member of retinal determination gene network (RDGN), which regulated cell proliferation, apoptosis, tumor growth and progression , 10. In The chemokine (C-X-C motif) ligand 1 (CXCL1) belongs to CXC chemokine family. CXCL1 was regulated by multiple signal pathways and tumor microenvironment. For example, tumor necrosis factor (TNF) and vascular endothelial growth factor (VEGF) stimulated CXCL1 expression via JNK, p38 MAPK, and PI-3K/Akt signaling pathways in human lung carcinoma epithelial cells , 25. CXCThe present study investigated the association between the expression profiles of CXCL1 and the clinicopathological features, as well as the prognostic value of CXCL1 in NSCLC. Besides, we also explored the relationship between DACH1 and CXCL1 in NSCLC.Human lung cell lines (A549 and SKLU-1) were originally obtained from ATCC  and we acquired from Dr. Guoan Chen in University of Michigan . These wTwo commercially available tissue microarray (TMA) slides  were purchased for immunohistochemistry (IHC). BC041115c, with core diameter of 5 mm containing 40 cases of squamous cell carcinomas, 48 adenocarcinomas, 3 adenosquamous carcinomas, 4 bronchioloalveolar carcinomas, 3 large cell carcinomas, 8 small cell carcinomas, 4 lung atypical carcinoids and 10 normal lung tissues was used to detect the expression of CXCL1. HLug-Ade150Sur with 75 matched pairs of human lung adenocarcinoma and adjacent lung tissues was used to evaluate the prognostic value of CXCL1 based on the detailed survival data. In addition, the protein level of DACH1, cyclin D1, Ki67, and CXCL1 in A549-vector and A549-DACH1 tumor tissues from xenograft mice models were examined by IHC. The specific primary antibodies include anti-CXCL1-antibody (ProteinTech), anti-DACH1-antibody (ProteinTech), anti-Ki67-antibody (Abcam), and anti-cyclin D1-antibody (MAIXIN-Bio). IHC staining was performed by Bios Biotech, Inc., with a 2-step protocol as previously described . Slide iFor quantification, at least three fields at 200× magnification of each spot were selected for IHC scoring. The immunohistochemical score were assessed independently by two experienced pathologists without knowledge of patients' characteristics. Scores were calculated on intensity and percentage of positive staining tumor cells in the whole tissue stains according to the Fromowitz Standard as described . BrieflyR2 = 0.9947.To evaluate the serum concentration of CXCL1, we collected the blood samples from about 100 patients who were diagnosed as ADC at various stages by April 2015 in Tongji hospital. The clinical samples collection and protocols were approved by the ethics committee of the Tongji Hospital of Huazhong University of Science and Technology. Serum samples from patients with obvious symptom of inflammation were excluded. The ELISA kit for CXCL1 was purchased from Cloud-Clone Corp. Diluted samples, including standard CXCL1 content, negative control and serums, are pipetted into 96 well plate pre-coated with anti-CXCL1 antibody. All procedures were performed following standard protocols with manual. After stopping the reaction, optical densities (OD) were measured at 450 nm using BioTek microplate reader. The intensity of this signal is directly proportional to the concentration of human CXCL1 present in the standard specimen. Standard curve demonstrated the linearity CXCL1 in cell supernatant samples was measured using SearchLight proteome arrays . Briefly, the samples including A549-vector, A549-DACH1 and SKLU-1-vector, SKLU-1-DACH1 were diluted to 1:5, 1:50 or 1:1,000 before 1 h incubation on the array plate that was pre-spotted with capture antibodies specific for CXCL1. Plate was decanted and washed three times before adding a cocktail of biotinylated detection antibody to each well. After incubating with detection antibody for 30 min, plate was washed three times and incubated for 30 min with streptavidin-horseradish peroxidase. Plate was again washed before adding SuperSignal Femto Chemiluminescent substrate. The plates were immediately imaged using the SearchLight imaging system, and data was analyzed using Array Vision software.# AAH-CYT-3-4) was purchased to detect the CXCL1 level in the supernatant of lung cancer cell lines. The detected samples included cell-free supernatant of A549-vector, A549-DACH1, SKLU-1-vector, and SKLU-1-DACH1. The operation steps are carried out according to the protocol provided by the manufacturer, as previously described  databases for mRNA expression of CXCL1. We obtained these published datasets from ArrayExpress and Oncomine . And theP-value were calculated and plotted on the online Kaplan–Meier plotter . The background database, required from GEO, offers gene expression data and survival information. The Affymetrix ID for CXCL1 was 204470_at, which includes 2,435 cases of lung ADC and SQC. The follow up time was threshold at 150 months. The patients were divided by median expression of CXCL1. The Kaplan–Meier survival curves were downloaded from the website. And the blend curves were obtained from IBM SPSS Statistics 19.0 based on GSE31210. All curves were resized in Adobe Illustrator CS6.Kaplan–Meier survival curves with HR and log-rank t-test and one-way ANOVA and the significance level was set at 0.05. Univariate cumulative survival analyses for PFS and OS were calculated using Kaplan–Meier method with the log-rank test. The correlation analysis was calculated according to Person χ2-test. Statistical analyses were conducted using SPSS 19.0 and GraphPad Prism 6.0. All data were presented as the mean ± SD.Statistical analysis between groups were calculated by Student's We detected the expression of CXCL1 in normal and lung cancer tissues by IHC staining. The representative images of normal and cancer tissues were shown in P < 0.0001) (P = 0.0001), tumor size (P = 0.0032) and lymph node metastasis (P = 0.0234) (n = 32) and low (n = 39) subgroups. Medium OS time of the CXCL1 high and low subgroups were 30 ± 2.98 and 48 ± 2.14 months, respectively, which indicated that they had significant difference of survival  and CXCL1 expression  were prognostic factors for OS. Multi-various Cox analysis revealed that TNM stage  and CXCL1 expression  were independent prognostic factors for OS.Considering the high protein abundance of CXCL1 in lung adenocarcinoma, we further analyzed the expression of CXCL1 in a TMA containing lung adenocarcinoma with matched adjacent lung tissue and clinicopathological parameters. Representative images of IHC staining for adjacent tissues and adenocarcinoma were shown in  0.0001) . The pro 0.0234) . HoweverR2 = 0.9947          or ADC ( = 0.0%)  or SQC ( = 0.0%) . However= 33.6%) . Further = 0.0%)  and ADC  = 6.0%) , but not = 0.0%) . There w = 0.0%) .R = 0.684, P = 0.042; R = 0.222, P = 0.006). To test whether DACH1 directly regulated CXCL1, two lung cancer cell lines A549 and SKLU-1 were transferred with control or DACH1 plasmid, respectively. We collected the supernatant of A549-vector, A549-DACH1, SKLU-1-vector and SKLU-1-DACH1 cell lines and detected the protein level of CXCL1 by SearchLight proteome array. The results demonstrated that DACH1 significantly reduced the expression of CXCL1 in A549-DACH1 and SKLU-1-DACH1 cells (Our previous study has demonstrated that DACH1 was a tumor suppressor factor and negatively correlated with CXCL5 . And CXCH1 cells . In addiH1 cells .P < 0.001) obviously decreased the expression of CXCL1 (P < 0.001), cyclin D1 (P < 0.001), and Ki67 (P < 0.001), suggesting that DACH1 not only reduced the expression of CXCL1, but also inhibited proliferation biomarkers cyclin D1 and Ki67 in vivo at protein level.Previous study showed that ectopic expression of DACH1 inhibited tumor growth . IHC stakmplot.com, respectively. The Kaplan–Meier curves from univariate Cox regression showed that patients with higher mRNA expression of CXCL1 tended to have shorter median OS  (P = 0.66) (P = 0.023) (P = 0.0015)  . Neverth = 0.66) . As expe= 0.023)  and PFS  0.0015) . In orde 0.0015)  and PFS  0.0015) . The resin vivo (Accumulating evidence has proved that CXCL1 plays an important role in the development of various malignant tumors. CXCL1, as an inflammatory factor, participates in the recruitment of granulocytes . Tumor-din vivo . In ER-nin vivo .Through immunohistochemical analysis, we found that CXCL1 protein expressed in ADC was higher than in other kinds of lung cancer. Therefore, we further evaluated the expression level of CXCL1 in ADC and its relationship with clinicopathological characteristics and prognosis. The analysis showed that the expression of CXCL1 in ADC was positively related to the TNM stage, tumor size and lymph node metastasis. CXCL1 was also positively associated with tumor grade, though it did not reach statistical significance. The survival curve plotted with survival information exhibited that the higher CXCL1 level accompanied with the shorter survival time. Previous study reported that circulating CXCL1 combining CCL18 could be as tumor markers for the differential diagnosis between ovarian cancer and benign ovarian masses . TherefoThe analysis results of 20 databases showed that mRNA CXCL1 positively associated with OS, PFS, TNM stage and lymph node metastasis for the ADC patients. But for the SQC patients, higher mRNA CXCL1 only related with shorter OS. Therefore, although the mRNA level of CXCL1 in SQC is higher than that of in ADC, the functions of CXCL1 were more obvious in ADC, suggesting that CXCL1 protein abundance and functions were also regulated by other factors.In vitro study confirmed that secretion of CXCL1 protein was significantly suppressed by DACH1 in lung cancer cell lines A549 and SKLU-1. Our previous study has demonstrated that DACH1 overexpression remarkably inhibited the growth of lung cancer in nude mice (in vivo. Previous finding demonstrated that tumor derived CXCL1 promoted the growth of lung cancer (As previous studies proved that DACH1 was considered as a tumor suppressor in several kinds of cancers including lung cancer and regulated multiple cytokine expression , 11, 14.g cancer , our stug cancer , 25. Ourg cancer . CXCL1, Our previous study showed that DACH1 antagonized the function of CXCL5 and revealed opposite effect on prognosis . To explAll datasets generated for this study are included in the article/Supplementary Material.The studies involving human participants were reviewed and approved by the ethics committee of the Tongji Hospital of Huazhong University of Science and Technology. The patients/participants provided their written informed consent to participate in this study.SY performed the experiments and drafted the manuscript. LX, MY, and SQ prepared the figures and tables. AL and KW designed the study and revised the manuscript. All authors contributed to this manuscript, read, and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.