Chlorella sp. isolated from the desert environment in Qatar, as a potential feed supplement. We found that the local strain showed a rich biochemical profile, with high amounts of proteins and lipids having biomolecules essential for human. Furthermore, the hexane extracts of this strain showed high antiproliferative activity against leukemia, one of the deadliest cancers in the world, proving its potential in the path of drug discovery against cancers. Our research findings thus highlight the nutraceutical potential of the local Chlorella sp., which can resist high temperatures of up to 40 °C, making it suitable for a large-scale production in ponds, year-round, for use as a sustainable food supplement.The world population is increasing rapidly, putting pressure on the existing resources, especially in the food sector. Besides having a continuous supply of food, it is also important to identify food sources that are nutritionally beneficial to the health of animals, fish, and humans. Hence, a balanced feed supplement including all the essential nutrients, such as amino acids, omega fatty acids, sugars, and carotenoids, is required. In this context, we have studied the beneficial effects of using a microalga, Chlorella, QUCCCM3, from the Qatar University Culture Collection of Cyanobacteria and Microalgae, was investigated for its growth characteristics and metabolite compositions for use as potential feedstock for food production. The strain was cultivated at 30, 35, and 40 °C, covering the annual average low and high temperatures in Qatar. The highest growth rates were recorded for cultures at 30 °C with 0.64 ± 0.04 day−1, followed by a growth rate of 0.54 ± 0.06 day−1 at 40 °C, indicating its thermotolerance ability. The biomass exhibited a high protein content (43 ± 2.3%), with existence of lysine (4.13%) as an essential amino acid, and docosahexaenoic acid, linoleic acid, and alpha-linolenic acid as important omega fatty acids present. On the other hand, Chlorella sp. QUCCCM3 also exhibited a high capacity for scavenging free radicals with an antiproliferative effect against chronic myeloid leukemia K562 cancer cells. The results indicate that Chlorella sp. QUCCCM3 is a promising candidate that can be produced year-round, in the Qatar environment, for commercial applications such as feed and nutraceutical supplements.Microalgae isolated from the Qatari desert was identified as thermotolerant, with a rich metabolite profile that is appropriate for use as food and health supplements. In this research, a species of  Microalgae biomass is highlighted as a promising resource for different applications. Energy industries are exploring their potential as biodiesel, bioethanol, and biogas . HoweverMicroalgae are mini factories, synthesizing important metabolites, such as proteins, carbohydrates lipids, essential amino acids, carotenoids, polyunsaturated fatty acids, and essential vitamins. These biological components are gaining importance as sustainable sources of feed, to satisfy the nutritional demands of a growing population ,4,5. ThePatinopecten yessoensis (Jay) scallop larvae [Microalgae are already known to support all the growth stages for marine and land animals . When mip larvae ,12.Besides nutritional benefits, microalgae also have high potential for pharmaceutical applications. This is due to the antioxidant properties of PUFAs and another class of secondary metabolites, such as carotenoids, found in microalgae. Omega-3 fatty acids and carotenoids are known for their health benefits in cases related to cancer and cardiovascular diseases . The comArthrospira sp., Dunaliella salina, Chlorella sp., and Tetraselmis sp. [Chlorella sp., are known to produce high amounts of proteins and other molecules with antioxidant properties, such as omega-3 and omega-6 fatty acids [Chlorella biomass has a positive influence on the animals when used as feed, due to its biochemical composition, palatability, and higher digestibility of nutrients. Similar positive impacts were recorded in aquaculture when the biomass was used as feed. These factors have favored their economical use as potential animal-feed constituents [The common species of algae used as food supplements include lmis sp. . Among tty acids ,18. Sevetituents .Chlorella sp. QUCCCM3, isolated from the local desert environment, was evaluated for its thermotolerance capacity, biomass productivity, metabolites content, antioxidant capacity, and antiproliferative properties. The strain was thus characterized to determine its suitability as feed component, to improve the health and nutritional value of the animals.Qatar is a peninsula that is governed by a hot and humid climate for most of the year. Food security and self-sufficiency in food products is of prime importance in the country. In our current research, a Chlorella sp. QUCCCM3, a freshwater microalgae, isolated from Qatar’s Desert environment, belonging to the Qatar Culture Collection of Cyanobacteria and Microalgae [Chlorella sp. QUCCCM3 was studied by using light microscopy , under 100× magnification. The strain was observed for its features, under two different abiotic stress, namely (i) continuous light and (ii) high temperature (40 °C). Culture at 30 °C and a photon flux density of 200 µmol photons m−2 s−1 with 12:12 dark: light cycles were used as control.croalgae  was seleChlorella sp. QUCCCM3 was cultivated by using BG11 freshwater growth medium. The composition of the medium was as follows: NaNO3 (1500 mg L−1), K2HPO4·3H2O (40 mg L−1), MgSO4·7H2O (75 mg L−1), CaCl2·2H2O (36 mg L−1), Na2CO3 (20 mg L−1), citric acid (6 mg L−1), Ferric ammonium citrate (6 mg L−1), EDTA (1 mg L−1), H3BO3 (2.86 mg L−1), MnCl2·H2O (1.81 mg L−1), ZnSO4·7H2O (0.222 mg L−1), CuSO4·5H2O (0.079 mg L−1), Na2MoO4·2H2O (0.390 mg L−1), and Co(NO3)2·6H2O (0.049 mg L−1) [ mg L−1) ,22. All Chlorella sp. QUCCCM3 was used to inoculate 5 mL of BG11 liquid media, which was incubated for 7 days at 30 °C, under an agitation of 200 rpm and a photon flux density of 200 µmol photons m−2 s−1, in an illuminated shaker . The culture was gradually scaled up to a volume of 500 mL and incubated under the previously mentioned conditions of growth, with an initial optical density at 750 nm (OD750nm) of 0.2. A daily assessment of the OD750nm was performed, using a spectrophotometer , for which 1 mL of the culture was taken and measured against a blank of only media without the cells. All cultures were done in duplicate, with two flasks, in the same experiment. The culture was harvested after 12 days of cultivation, at the onset of the stationary phase. The biomass was harvested and then freeze-dried prior to being subjected to assessment of its metabolite content. A single colony of Dry weight was determined in replicates , for whiT1 is the time corresponding to the beginning of the exponential phase; T2 is the time corresponding to the end of the exponential phase; N1 is OD750nm at T1; and N2 is OD750nm at T2.The growth rate (µ) was determined by using the following formula :µ=lnN2−lX2 is biomass concentrations (g L−1) at Tend (end point of culture), and X1 is the biomass concentration (g L−1) at Tinitial (start point of cultivation). All measurements were performed in triplicates.Biomass productivities were calculated based on biomass dry weight, using the following formula :P=. Set points were 2.00, 1.244, and 2.00 μmol photons·s−1 for channels A, B, and C, respectively, fixed for a photon flux density of approximately 200 µmol photons m−2 s−1, which is considered to be optimum for the standard cultivation of microalgae, at 12 h:12 h dark:light cycles. A daily assessment of the OD750nm was performed to be able to compare the growth performance of the strain under the different temperatures tested.For the study of the thermotolerance capacity, escribed , to 1 L The microalgal biomass collected after 12 days of cultivation, corresponding to the end of exponential phase of culture, was washed with deionized water and freeze-dried  prior to being subjected for metabolite analysis. For total protein, 25 mg of dried microalgae was hydrolyzed overnight at 60 °C, using 5 mL sodium hydroxide (NaOH 0.1 M) from Sigma-Aldrich  . The totg for 5 min, and supernatant was transferred into a pre-weighed tube. The methanol chloroform extraction step was repeated to ensure complete removal of lipids from the biomass. The organic phase was separated from the aqueous phase by adding 0.88% of NaCl and mixing of the solution. The organic phase was collected, dried, and weighed, and the lipid content was measured gravimetrically [Total lipids were extracted from freeze-dried algae biomass, using the method of Folch, with some modifications, where freeze-dried biomass was treated with sodium chloride solution (0.88%) and incubated at 4 °C, in an adequate volume of methanol. Post overnight incubation, double the volume of chloroform  was added to the mixture, and cells were disrupted by tissue lyzer . The mixture was centrifuged at 5000× trically ,29. The trically .Lipid cCarbohydrates were estimated using by phenol sulphuric acid reagent . For theAmino acids were quantified by using pre-column derivatization with O-phtalaldehyde (OPA) and 9-fluorenylmethyl chloroformate (FMOC) after methods as described in Reference . Pre-wei2SO4:CH3OH = 1:10), and sonicated for 10 min. This step was followed by heat treatment at 80 °C for 2 h. Further, the mixture was transferred to a tube containing 1 mL of distilled water and 3 mL of hexane: chloroform  mixture and centrifuged. The top layer containing FAME fractions was filtered and analyzed, using GD-FID  [FAMEs were extracted via a one-step transesterification method, where a known amount of freeze-dried biomass was treated, using sulfuric acid  and methanol solution  . Then, 2−2 s−1, with continuous light, 24:00 (day and night cycle), using an illuminated shaker . Here, the only stress affecting the biomass was continuous light. The optical density of the culture was monitored at 750 nm, and the culture was harvested at the late stationary phase. Algae secondary metabolites (carotenoids) were extracted via maceration, using hexane  as an organic solvent [The microalgae culture was cultivated at a scale of 500 mL, using BG11 growth media. The strain was cultured at 30 °C, representing the low average annual temperature in Qatar and optimum temperature for best microalgal growth. The mixing was set to 150 rpm, under a photon flux density of 200 µmol photons m solvent . For the solvent :Caroteno−1. The crude extract was stored at 4 °C, in the dark, prior to being used for the investigation of its antioxidant and antiproliferative capacities.Further, the extract was dried, weighed, and dissolved in 1% dimethylsulfoxide (DMSO), to a concentration of 100 mg mLThe Trolox Equivalent Antioxidant Capacity (TEAC) assay was performed by using the Sigma-Aldrich TEAC Kit , in which a ferryl myoglobin radical is formed from metmyoglobin and hydrogen peroxide, which oxidizes the 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), to produce ABTS+, a cation that is green in color and determined spectrophotometrically at 405 nm, using Synergy H4 Hybrid Multi-Mode Microplate Reader . A blank was prepared consisting of all the reagents except the algal extract to auto zero any absorbance caused by the reagents.The assay reflects the ABTS+ radical-scavenging capacity, in which a decrease in absorbance was observed due to the antioxidant molecules in the extract tested. All work was performed in the dark, at room temperature ,38.−1 streptomycin, and 2 mM l−glutamine. All the reagents were obtained from Sigma-Aldrich, St. Louis, MO, USA. Cultures were maintained in a CO2 incubator  at 37 °C. Cell viability was tested by using trypan blue dye exclusion method. The effects of Chlorella sp. QUCCCM3 extracts on proliferation of leukemia cancer cell line K562 CML (Chronic Myelogenous Leukemia) were determined by using colorimetric micro-culture assay with the 3--2,5-diphenyl-2H tetrazolium bromide (MTT) as end point [−1), for an incubation period of 24 h. An additional well was prepared as a blank, containing 1% DMSO, allowing a final concentration of 0.1%. Such a concentration was previously described as safe for the cells [Human chronic myeloid leukemia K562 cells from ATCC were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 IU/mL penicillin, 100 μg mLnd point . AntiproChlorella sp. QUCCCM3 crude extract. The standard error of mean values was calculated at p < 0.05 level of significance.Data were expressed as the mean of two independent parallel experiments. One-way ANOVA was used to assess the differences amongst the biomass productivities obtained after the different treatments and also between the inhibition % of the leukemia cancer cells from K562, using the different concentrations of the Chlorella sp. QUCCCM3 was cultivated under different temperatures, namely 30, 35, 40, and 45 °C, knowing that 30 °C corresponds to the annual average temperature, and 40 °C to the high annual average temperature, in Qatar. Chlorella sp. QUCCCM3 presented a high growth rate of 0.64 ± 0.04 day−1 and a biomass productivity of 0.132 ± 0.01 g L−1 day−1, as shown in Chlorella sp. QUCCCM3’s growth decreased slightly with a temperature increase up to 40 °C; however, it exhibited identical values of ~0.54 °C day−1 at 35 and 40 °C. Similarly, the biomass productivity at 30 °C was significantly higher (p < 0.5) than those recorded for the cultivation at 35 and 40 °C, showing similar biomass productivities of 0.118 ± 0.02 g L−1 day−1 and 0.111 ± 0.01 g L−1 day−1, respectively (p > 0.05), whereas no growth was recorded for the strain when cultivated at 45 °C. Our results proved that Chlorella sp. QUCCCM3 is a fast-growing strain that can adapt at high temperatures, up to 40 °C, corresponding to the high average temperature in Qatar. This confirms its thermotolerance capacity and therefore its suitability to be cultivated year-round, at a large scale, under Qatar’s harsh climate.Chlorella sp. QUCCCM3 presented a high amount of proteins, reaching up to 43 ± 2.3%, whereas the lipid amount was found to be 33 ± 1.75%. Additionally, the carbohydrates estimated was up to 13.5 ± 0.5%, as illustrated in An assessment of the metabolites content was performed for QUCCCM3, to investigate its nutritional potential. The biomass obtained at 30 °C was subjected to quantitative and qualitative metabolite analysis. Results showed that Chlorella sp. QUCCCM3 was analyzed through amino acid profiling. The obtained data are illustrated in To support these findings, the quality of proteins in Chlorella sp. QUCCCM3 was also determined, and the results are presented in −1 representing 35% of the total fatty acids. In addition to the amino acids, the FAME profile of Chlorella sp. QUCCCM3 showed a high growth rate and interesting biochemical profile, evidencing its suitability to be considered as a promising candidate for food supplement production. To further enhance its nutritional quality by increasing the amount of high-value added compounds associated with multiple health benefits, two different abiotic stressors, namely high temperature (40 °C) and continuous light, were applied separately. The comparative analysis of the cell morphologies when cultivated under both stresses, along with control condition, highlighted differences in the cell shape, size and, contents . The hexane extract of Chlorella sp. QUCCCM3 showed an IC50 value of 21.37 ± 2.98 μg mL−1 , Chlorella sp. QUCCCM3 was stable up to 40 °C, with negligible variations in growth. Therefore, the strain is considered thermotolerant, as it can grow in higher temperatures, as compared to other known Chlorella strains. Its tolerance can be attributed to its origin of isolation and its evolutionary adaptation to a desert climate over the years, making it unique. Our findings also showed best biomass productivity of 0.132 ± 0.01 g L−1 at 30 °C for Chlorella sp. QUCCCM3, which was higher than the productivity recorded for other Chlorella spp. (0.114 ± 0.850 g L−1 day−1), as reported by different researchers [The growth rate obtained for 66 day−1 . Howeverat 38 °C . It is kat 38 °C ,41. It wearchers , the optearchers . It shouearchers ,45.Chlorella sp. QUCCCM3, demonstrated its capacity for high growth and biomass production under favorable cultivation conditions, thereby proving its commercial suitability. Chlorella sp. QUCCCM3 further exhibited a biomass concentration value of 1.72 ± 0.01 g L−1, which slightly decreased at 35 and 40 °C, although the cells still expressed normal growth with the rise in temperature, which is remarkable. A similar trend was confirmed by several other studies on Chlorella sp. [Microalgae are an important source for feed supplements and bioactives ,47. Howeella sp. ,50.Chlorella sp. QUCCCM3, 43 ± 2.3%, was higher than commercially available Chlorella sp., having a value of 39.98% [Chlorella sp. QUCCCM3 was enriched with all the essential amino acids with the existence of Lysine up to 5%, an important amino acid, indicating its suitability as potential feed. This amino acid also imparts a sweet flavor to the biomass, to improve the palatability for livestock, which is an important criterion for feed supplement production [Chlorella sp. QUCCCM3 exhibited high amounts of glutamate, thus highlighting the sensory characteristics of the microalgae and improving the taste, when used as food ingredient [In addition to the biomass productivity, metabolite production is the other imperative factor toward commercial utilization of microalgae for nutritional purposes . The prof 39.98% . Proteinoduction . Similargredient .Chlorella spp. ranged from 28 to 32% [Chlorella sp. QUCCCM3 exhibited a slightly higher amount of lipids when compared to the given range of values. Precisely, the lipid content in Chlorella sp. QUCCCM3 was found to be much higher when compared to the amounts observed for Chlorella pyrenoidosa and Chlorella vulgaris which presented values around 2% and 14–22%, respectively [Chlorella sp. QUCCCM3 biomass proved its commercial potential for producing feed for aquaculture [Chlorella sp. QUCCCM3 also possesses LA, which is observed in high amounts in corn oil, sunflower oil, walnuts, brazil nuts, and soya beans, and hence can be used to replace the oils and provide the same nutritional benefits, without causing the side effects, such as cardiovascular diseases. Palmitic acid found among the FAMEs is known to improve the quality of milk by regulating milk fats [Chlorella sp. QUCCCM3 has the potential to be used as feed for livestock production. A similar trend was seen for Rhodophytas, which also exhibited high palmitic acid content. However, the amount was lower than the values found in microalgae [Chlorella sp. QUCCCM3 isolate from Qatar showed slight variations in its FAME profile, as compared to strains found in Australia [Chlorella sp. QUCCCM3. Our local microalgae strain exhibited an omega-6/omega-3 ratio of 0.42, which is within the recommended range (0.25–1) for this strain to be beneficial also for human use [Chlorella sp. QUCCCM3 exhibits the cellular mechanisms for producing these compounds and thus can be modulated through variation of the cultivation regime [In regard to the total lipids, the values reported for all 8 to 32% . Chlorelaculture . It was aculture ,58. Besiaculture . Furtherilk fats ,12. In tcroalgae . The Chlustralia . While tuman use . The pren regime ,61.Chlorella sp. QUCCCM3, it was within the range that is commonly seen for microalgae [With regard to carbohydrate content of croalgae .Chlorella sp. QUCCCM3 was subjected to abiotic stress, to enhance its nutraceutical potential. Morphological differences, such as changes in cell size and shape, were observed under both stresses applied separately. The interesting observation that was made about the cell size at 40 °C can be explained by a phenomenon called gigantism that was previously described for Chlorella sp. under temperature stress [e stress . MoreoveChlorella sp. QUCCCM3 microalgae also exhibited large circular vesicles of carotenoids under continuous light stress, as seen in Chlorella sp. QUCCCM3 when cultivated under normal conditions (control) and at 40 °C. It has been proved widely that light is a key factor influencing the production of carotenoids within the cells. The accumulation of these pigments can be attributed to photo-induced activation of gene-encoding enzymes involved in the β-carotene biosynthesis [Chlorella sp. QUCCCM3 was found to be higher than the value reported in other research using different Chlorella sp., exhibiting 65.2 ± 2.5 μmol g−1 and 56.09 ± 1.99 μmol g−1, respectively [Chlorella spp., as reported for Chlorella protothecoides (3.49 ± 0.13 μmol g−1), Chlorella pyrenoidosa (8.48 ± 0.42 μmol g−1), Chlorella vulgaris (5.53 ± 0.05 μmol g−1), and Chlorella zofingiensis (1.83 ± 0.21 μmol g−1) [Chlorella sp. QUCCCM3 as a nutraceutical supplement. The health benefits of these molecules are well-known, specifically as anticancer molecules [Chlorella sp. QUCCCM3 crude extract was also carried out, and the results showed an interesting IC50 below the standard limit described by the American National Cancer Institute (IC50 ≤ 100 μg mL−1). It has been recommended that lower the IC50 value, the higher the potential of the molecules for drug development is. Furthermore, this activity is about half of that described previously for C. ellipsoidea and C. vulgaris extracts, showing IC50 of 40.73 ± 3.71 and 40.31 ± 4.43 μg mL−1, respectively [Chlorella sp. QUCCCM3 is naturally adapted to produce secondary metabolites, such as carotenoids, to protect the cells when exposed to abiotic stressors common throughout the harsh Qatari climate. This also converges with other findings proving that microalgae, when under stress, developed components for survival and defence unique to them, in comparison to the terrestrial plants [Chlorella sp. QUCCCM3 crude extract showed a peak of absorption at 480 nm, indicating the presence of carotenoids [ynthesis . The premol g−1) . The pigolecules . For thiectively . This inl plants . Chlorelotenoids .Chlorella sp. QUCCCM3 demonstrated unique characteristics and can be considered for year-around cultivation due to its thermotolerance capacity. In addition, the presence of high protein content with essential amino acids with high amounts of glutamate and lysine confirmed its nutritional potential. PUFAs, such as DHA and carotenoids, found in the local strain serve as powerful antioxidants. Furthermore, the extracts from the strain also have significant anticancer activity against Leukemia cancer cells, making them interesting from a pharmaceutical perspective. Therefore, Chlorella sp. QUCCCM3 can be considered to be a promising alternative that has potential for sustainable food production in an arid environment.


The apolipoprotein E (ApoE) gene polymorphism has been found to influence plasma lipid concentration, and its correlation with peripheral arterial disease (PAD) has been investigated. However, it is unclear whether ApoE is associated with PAD in Chinese type 2 diabetes mellitus (T2DM) patients. Therefore, our study is aimed at investigating the relationship between the ApoE gene polymorphism and PAD in Chinese T2DM patients.  A total of 192 T2DM patients were divided into two groups: T2DM and T2DM with PAD. The clinical and biochemical parameters were obtained. Polymerase chain reaction was used to identify the genotypes of ApoE. The multivariable logistic regression analysis was used to identify the possible risk factor for PAD. ε4/ε4/ There were no significant differences in the genotype and allele frequencies of ApoE between the T2DM and T2DM with PAD groups. However, the T2DM with PAD group tended to have more  These results demonstrated that there was no evidence of a relationship between ApoE and PAD. Diabetic patients with peripheral arterial disease (PAD) are at higher risk of increased morbidity compared to healthy people . PAD is ε2, ε3, and ε4), and these alleles in turn form six different ApoE genotypes  [ε4 allele better corresponds with higher cholesterol levels and vascular disease risk than the ε2 allele and the ε3 allele [ε2ε2 genotype was associated with an increased risk for PAD in patients at high risk for cardiovascular disease [Previous studies have shown that the prevalence of PAD increases with age, smoking, and diabetes , 6. Reced ε4/ε4) . General3 allele , 14. Mul3 allele . However disease .To date, no studies have evaluated the association between ApoE and PAD in Chinese type 2 diabetes mellitus (T2DM) patients. Therefore, this study is aimed at investigating the possible effect of ApoE on PAD in Chinese T2DM patients.The study protocol had been approved by the Ethics Committee of Changzhou No.2 People's Hospital. Written or verbal consent forms were obtained from each patient, and the motive of the study was clearly explained to them. A total of 192 T2DM patients were recruited from Changzhou No.2 People's Hospital from January 2017 to October 2017. T2DM was defined as fasting plasma glucose ≥ 7.0 mmol/L or 2 h oral glucose tolerance test ≥ 11.1 mmol/L according to the criteria of the World Health Organization . 122 pat2 mL of fresh blood sample was taken from each patient and collected in EDTA-containing tubes. Genomic DNA was extracted from peripheral blood using the DNA extraction kit  and amplified by polymerase chain reaction (PCR). The primers for this reaction were sense 5′ AACAACTGACCCCGGTGGCG 3′ and antisense 5′ ATGGCGCTGAGGCCGCGCTC 3′ . PCR begBriefly, the ankle-brachial index (ABI) was derived from the calculation of the ratio of the ankle systolic blood pressure (SBP) divided by the arm SBP . The ankBlood samples were collected from the patients after 10-12 h overnight fast and then immediately determined within 1 h. Total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were measured by an automated analyzer . Glycated hemoglobin (HbA1c) values were determined by the high-performance liquid chromatography method .t-tests were performed to find any differences in the age, disease duration, BMI, HbA1c, TC, HDL, LDL, and TG between the T2DM and T2DM with PAD groups. Categorical data, such as sex, hypertension, family history of diabetes, and smoking habits, were analyzed between the two groups by the chi-squared test or Fisher's exact test. The frequency differences of the ApoE allele and genotype between groups were tested by chi-squared tests or Fisher's exact test. The adjusted odds ratio (OR) with 95% confidence interval (CI) was used to determine the independent risk factor for PAD by multivariate logistic regression analysis after adjusting for age, sex, BMI, TC, TG, HDL, LDL, HbA1c, smoking habits, hypertension, disease duration, family history of diabetes, and ApoE. Statistical significance was defined at P < 0.05.All statistical analyses were performed with SPSS 16.0 . Data from normally distributed parameters are presented as the mean ± SD. Independent sample Initially, a total of 240 Chinese T2DM patients participated in the present study. 48 subjects were excluded due to uncompleted questionnaires, unsuccessful genotyping, or both. Finally, 192 participants were included in the sample analysis. The demographic and biochemical data of the patients are shown in ε4/ε3 genotypes (21.5% vs. 11.3%) than the T2DM group. Also, the ε4 allele was more frequent, and the ε2 allele was less frequent in the T2DM with PAD group, but the differences were not statistically significant.The distributions of the ApoE genotypes and allele frequency are shown in Tables ε3/ε3 carriers (ε3/ε3), ε4 carriers (ε3/ε4 and ε4/ε4), and ε2 carriers (ε2/ε3 and ε2/ε2). Two participants who had the ε2/ε4 genotype were excluded from this analysis because of their opposing effect on lipid metabolism. There were no significant differences in plasma lipid levels among three ApoE allelic groups. Additionally, we did not find significant differences in the lipid profile between ε3/ε3 carriers, ε4 carriers, and ε2 carriers in either group.The relationship between ApoE alleles and plasma lipid levels is shown in Tables ε2/ε2 and ε3/ε2), 2 = ApoE3 (ε3/ε3 and ε4/ε2), and 3 = ApoE4 (ε4/ε3 and ε4/ε4)), BMI, TC, TG, HDL, LDL, HbA1c, and disease duration were considered as covariates. The results are shown in P = 0.006, OR = 2.710, 95%CI = 1.325‐5.541 (smoking); P = 0.000, OR = 1.067, 95%CI = 1.032‐1.105 (age); P = 0.036, OR = 1.070, 95%CI = 1.005‐1.141 (disease duration); P = 0.013, OR = 1.593, 95%CI = 1.103‐2.301 (TG); P = 0.001, OR = 2.073, 95%CI = 1.338‐3.213 (LDL); and P = 0.029, OR = 0.830, 95%CI = 0.703‐0.981 (HbA1c)).The multivariate logistic regression analysis was used to analyze the clinical factors correlated with PAD, all subjects were considered as a whole, and the presence of PAD was considered as a dependent variable (1 = PAD and 0 = non − PAD); sex , hypertension (1 = hypertension and 0 = nonhypertension), smoking (1 = smoker and 0 = nonsmoker), family history of diabetes (1 = family history of diabetes and 0 = no family history of diabetes), ApoE (1 = ApoE2 (ε4/ε3 genotypes (21.5% vs. 11.3%) than the T2DM group. Further studies are needed to explore the potential role of ApoE in PAD in Chinese T2DM patients.In our study, there was no relationship between ApoE and PAD in Chinese T2DM patients. However, the T2DM with PAD group tended to have more ε4 allele is associated with a high level of serum TC and LDL in the Chinese population. To demonstrate that the ε4 allele has a great influence on the lipid levels, we analyzed the plasma lipid levels among three ApoE allelic groups and between ε3/ε3 carriers, ε4 carriers, and ε2 carriers in both groups, respectively. Although ε4 carriers had higher levels of LDL than ε3/ε3 and ε2 carriers, the differences were not statistically significant. This might be due to the differences in genetic background and dietary restrictions and lipid-lowering aggressive treatment in the patients.The association of ApoE and the lipid profile remains controversial. The ε2/ε2 genotype seemed to be related to both mild and severe PAD [ε4 carriers was observed [ε4/ε3 group with newly diagnosed and prevalent diabetes had a significantly higher prevalence of PAD than other diabetic subjects among ever-smokers [ε4/ε3 than the T2DM patients, but this did not reach statistical significance. This lack of significance might be due to the relatively small number of patients and the influence of ethnic differences. It is clear that the frequencies of ApoE allele and genotype vary between different populations [In this study, PAD refers to arterial occlusive disease of the lower and upper limbs , and itsvere PAD . In a soobserved . In a Ja-smokers . In our ulations , 21. TheThe traditional risk factors  are produced by glucose, protein, and lipid through nonenzymatic glycosylation reactions , so HbA1In terms of limitations, our study population is limited to T2DM patients. As such, our results might not be applicable to healthier individuals. In addition, this study is limited to the Chinese sample size. Thus, the genetic association found in this study might not be generalized to other ethnic populations. Finally, we did not collect data on drug therapy, and we plan to collect the above data in our further study.In summary, our study found that there was no relationship between ApoE and PAD. Moreover, smoking, age, disease duration, TG, and LDL can aggravate the progression of both diseases. Furthermore, T2DM patients identified to carry high-risk factors should be treated aggressively to prevent the progression of PAD.


Men and women are sexually dimorphic but whether common anthropometric and biochemical parameters predict type 2 diabetes (T2D) in different ways has not been well studied. Here we recruit 1579 participants in Hainan Province, China, and group them by sex. We compared the prediction power of common parameters of T2D in two sexes by association, regression, and Receiver Operating Characteristic (ROC) analysis. HbA1c is associated with FPG stronger in women than in men and the regression coefficient is higher, consistent with higher prediction power for T2D. Age, waist circumference, BMI, systolic and diastolic blood pressure, triglyceride levels, total cholesterol, LDL, HDL, fasting insulin, and proinsulin levels all predict T2D better in women. Except for diastolic blood pressure, all parameters associate or tend to associate with FPG stronger in women than in men. Except for diastolic blood pressure and fasting proinsulin, all parameters associate or tend to associate with HbA1c stronger in women than in men. Except for fasting proinsulin and HDL, the regression coefficients of all parameters with FPG and HbA1c were higher in women than in men. Together, by the above anthropometric and biochemical measures, T2D is more readily predicted in women than men, suggesting the importance of sex-based subgroup analysis in T2D research. T2D is currently diagnosed by fasting plasma glucose (FPG), hemoglobin A1c and the oral glucose tolerance test (OGTT)3. But these factors remain insufficient in predicting the development of diabetic complications5. Therefore, efforts have been focused on identifying better predictors for T2D. Many anthropometric and biochemical markers have been investigated, including BMI, body adiposity index (BAI), waist circumference and waist-to-hip ratio, waist-to-height ratio, high blood pressure, triglyceride levels, insulin and proinsulin levels, and proinsulin-to-insulin ratio (P/I ratio). However, some studies remain inconsistent and conclusive results have not been reached.High plasma glucose levels can cause diabetes mellitus, which can lead to clinical complications including cardiovascular morbidities, renal impairment, retinopathy. Type 2 diabetes (T2D) can result from the dysfunction of insulin-secreting β-cells, the resistance of insulin by peripheral tissues such as muscle, or both6. T2D is one of the most prevalent diseases that also show sexual dimorphism in many aspects, including diagnosis, prevention, and treatment8. For example, men have a higher risk of T2D and are diagnosed at a younger age12. However, women with T2D are prone to develop more severe complications than men13. Women with T2D have a higher risk for coronary heart disease than men with T2D16. The sex difference in T2D has been attributed to both biological and psychosocial factors7.Men and women are different in many ways; however, other than a few diseases related to the reproduction system such as breast cancer and testicle cancers, the sex difference in many prevalent diseases has not been carefully evaluated17. In a pre-diabetes cohort of East Asians, age, FPG, triglyceride levels, and smoking status were found to be associated with T2D development stronger in men while waist circumference was found to be associated with T2D development only in women18. In multiple studies, men were diagnosed with T2D at a lower BMI21. HbA1c predicted T2D better in women in a French cohort22. Despite these interesting studies, a systematic comparison between the two sexes has not been reported.Several anthropometric and biochemical parameters are associated with T2D differently between men and women. For example, in a study on middle-aged Caucasians, low vitamin D3 was independently associated with T2DM in women but not in menIn this study, we analyzed a cohort of 1579 East and Southeast Asians from Hainan Province, China, comparing between women and men the association and prediction power of multiple anthropometric and biochemical parameters. Our study provides novel knowledge regarding the sex-difference in T2D prediction and prevention.2), smoking, hyperlipidemia, and hyperuricemia (Table The 1579 participants were grouped into 567 men and 1012 women. Men had slightly higher T2D cases (N = 93) compared to women (N = 128). Complications related to cardiovascular diseases and diabetic complications were rare Table . The casia Table , which wWe first compared the difference of multiple physical and chemical characteristics in males and females. The mean age of males was slightly older than females, with a mean ± standard deviation of 49.16 ± 13.03 vs 47.43 ± 13.5, respectively. Fasting plasma glucose was not significantly different between males and females . The percentage of HbA1c was slightly higher in males . Fasting and glucose-stimulated insulin levels did not differ significantly between males and females . Fasting and glucose-stimulated proinsulin were significantly higher in males . All other physiological and biochemical measurements showed a significant difference between the two sexes Table .Next, we were interested in knowing the sex difference regarding the correlation of HbA1c and FPG. As shown in Fig. We continued to examine other anthropometric and biochemical parameters for their correlation and regression pattern with FPG and HbA1c, as well as their prediction strength for T2D. As shown in Fig. As shown in Fig. Despite the generally weak prediction power, both systolic and diastolic blood pressure could predict T2D better in women than in men: the ROC-AUC values were 0.66 ± 0.03 P < 0.001) vs 0.59 ± 0.03 (P < 0.01) by systolic blood pressure and 0.60 ± 0.03 (P < 0.001) vs 0.54 ± 0.03 (ns) by diastolic blood pressure  vs 0.58 ± 0.03 (P < 0.05) by triglyceride, 0.63 ± 0.03 (P < 0.001) vs 0.53 ± 0.03 (ns), by total cholesterol, 0.62 ± 0.03 (P < 0.001) vs 0.52 ± 0.03 (P < 0.01) by LDL and 0.58 ± 0.03 (P < 0.01) vs 0.50 ± 0.03 (ns) by HDL  in women than in men Fig. A. AlthouWe also examined postprandial insulin and proinsulin levels and found no correlation to FPG and HbA1c Figure . Linear Based on the results from ROC-AUC analysis, we calculated the optimal cutoffs for all parameters in predicting T2D using Youden’s J statistics and compared the sensitivity, specificity, and Youden Index between men and women Table A. At the23. For example, osteoporosis, autoimmune diseases, and Alzheimer’s diseases are more prevalent in women26. Autism and some cancers such as stomach cancer, oesophageal cancer, and liver cancer have a higher prevalence in men27. Recently, the new concept of sex and gender medicine has begun to attract attention. This new concept proposes the separation of men and women in the diagnosis, prevention and treatment of diseases which are currently treated by ‘one-size-fits-all' approach, and believes that sex-based prevention measures and therapies would benefit patients of both genders6.The sex difference in disease results from a combination of human genetics, physiology, behavior, or anatomy. It is believed that nearly all human diseases are sexually dimorphic, manifesting in prevalence, age of onset, severity, or disease course28. In this study, we obtain a panel of the most common anthropometric and biochemical parameters from 1579 participants distributed across the Hainan Province, China, and systematically study the association with FPG, HbA1c and, T2D incidence. We focused on comparing the difference between males and females. Our results show that HbA1c, age, waist circumference, BMI, systolic and diastolic blood pressure, triglyceride, total cholesterol, LDL, HDL, fasting insulin, and proinsulin levels all predict T2D better in women than in men. To our knowledge, this is the first study of its kind systematically focusing on the sex difference in predicting T2D.Few studies to date have focused on the sex difference in T2D, especially the diagnostic and predictive parameters32. The link between blood uric acid and T2D remains unclear35. Different from the above studies, our results show that the predicting powers of vitamin D3 and uric acid are weak (ROC-AUC < 0.6). No difference in the ROC-AUC values between women and men was observed  > waist circumference (0.70) > systolic pressure (0.66) = triglyceride (0.66) = fasting proinsulin (0.66) > fasting insulin (0.64) > total cholesterol (0.63) > BMI (0.62) = LDL (0.62) > LDL (0.62) > diastolic pressure (0.60); in men, age (0.69) > fasting proinsulin (0.62) > fasting P/I ratio (0.61). There are several interesting interpretations from this result. First, these measures are mostly poor predictors for men but moderate predictors for women, arguing for the importance of sex-based subgroup analysis in evaluating predictors for T2D. Second, different from many previous studies focusing only on one or two factors and proposing BMI and P/I ratio as good T2D predictors, our study finds them to be much poorer predictors than many other measures including blood pressure and lipid profiles. Third, in women, waist circumference alone rather than BMI is a good T2D predictor (ROC-AUC > 0.70), which is somehow surprising but consistent with two previous studiesIn all, by thoroughly comparing the correlation, regression, and ROC analysis of common anthropometric and metabolic measures between men and women from a large cohort in Hainan China, we show that T2D is more readily predicted by these parameters in women than in man, suggesting the importance of weighing sex difference in T2D diagnosis and prediction.39.Subjects participating in this study are ethnically a combination of east Asian and southeast Asian distributing across cities and countryside of Hainan Province, China. This study includes 1579 men and women of different ages, sex across different socioeconomical status and education levels. Such information is collected based on a survey before admitting participants for the glucose tolerance test. The study was approved by the Ethical Committee of Hainan General Hospital and all participants gave written informed consent. All methods involving humans were carried out in accordance with relevant guidelines and regulations. Original data was published before2). Waist circumference was measured at the midpoint between the lateral iliac crest and lowest rib to the nearest 0.5 cm. Systolic pressure and diastolic pressure were obtained by a standard sphygmomanometer. Glucose stimulation was conducted by a standard protocol of a 2-h, 75-g oral glucose tolerance test (OGTT). Fasting was defined by no food uptake and drinks (except for water) for at least 10 h. Blood was then drawn at the fasting state and glucose-stimulated state. Blood glucose, HbA1c, triglyceride, total cholesterol, LDL, HDL, fasting insulin, and proinsulin were measured by standard protocols in the Medical Laboratory in Hainan General Hospital.Weight and height were measured on a mechanic scale and a rod mounted on the wall. Values for weight and height were kept to the nearest 0.1 kg and 0.5 cm, respectively. BMI was calculated by dividing weight (kg) by the square of height (m39. Data of all 1579 participants were grouped by sex. Association/correlation study was conducted by calculating Spearman’s rank correlation coefficient using SPSS software. Linear regression and binary regression analysis were conducted by using SPSS software. ROC graph and AUC values were derived from pROC package in Rstudio. To visualize the linear regression, data were first converted by log10 to obtain normally distributed data, then visualized by Rstudio using ggplot2 package. A small number of outliers were removed for visualization purpose.Original data were published beforeStatistical analysis was conducted using the default methods in IBM SPSS version 24. Briefly, Chi-square was used to compare the frequency of diabetic complications and other conditions including smoking between the men group and women group. A two-tailed, unpaired Student’s t-test was used to compared mean values of parameters. Association was analyzed by using the Spearman method. P < 0.05 was considered statistically significant.Supplementary Information


The optimized batch was further loaded with RSV and evaluated for in vitro and ex vivo permeation and for in vivo hepatotoxicity. The developed ITT-SNEDDS exhibited globule size below 300 nm, up to 272.27 ± 7.12 mcg/cm2.h and 61.27 ± 2.83% of steady-state flux (JSS) and permeability % respectively. Optimum formulation consisted of 0.15 g oil mixture, 0.6 g of surfactant (Labrasol), and 0.250 g co-surfactant (Transcutol). Permeability studies confirmed the enhanced permeation percentage of ITT (40.77 ± 1.18%), and RSV (29.94 ± 2.02%) from optimized formulation, with enhanced steady-state flux (JSS). In vivo studies demonstrated the superior hepatoprotective activity of optimized formulation compared to a different drug formulations and marketed product. Therefore, RVS loaded ITT-SNEDDS might be a successful strategy for acne management with improved action, and minimum side effects.Acne Vulgaris is one of the most common chronic inflammatory skin disorders that affect majority of teen-agers worldwide. Isotretinoin (ITT) is the drug of choice in the management of acne, but, it suffers from serious side-effects including hepatotoxicity, and some psychological disturbances following its oral intake. The objective of this study was to develop and optimize ITT loaded nanoemulsions (ITT-SNEDDS) and to incorporate resveratrol (RSV)in optimum formulation to decrease ITT side effects The ITT solubility was first tested in various essential oils, surfactants, and co-surfactants to select the essential nanoemulsion ingredients. Mixture design was applied to study the effect of independent variables and their interactions on the selected dependent responses. The developed ITT-SNEDDS were characterized for their globule size and  Food and Drug Administration) in 1982 to treat severe acne , chemically 3,4,5-trihydroxystilbene, available in two different forms,  Baxter, . RSV has Baxter, , anti-in Baxter, , cardio- Baxter, , anti-ox Baxter, , and ant Baxter,  effects. Baxter, . RSV has Baxter, . Drug-meThe potential application of RSV as a therapeutic agent has been limited due to photosensitivity and partial intestinal absorption, thus decreasing bioavailability and causing extensive elimination and catabolism through ABC transporters , on dilution, produce nano-sized transparent systems called nanoemulsions . Essential oils were obtained from Hayward, CA, USA. RSV, was procured from Water Import and Export Co., Ltd (China). Triglycerides, propylene glycol monolaurate, and glycerol monooleate, Labrasol, and transcutol were generously gifted by Gattefosse (France). All other reagents and chemicals used were of analytical grades.2.2.The solubility of ITT in various oils, surfactants, and co-surfactants was determined. An excess amount of ITT was dissolved in about 2 ml of each vehicle individually. The mixture was then shaken on a water bath  for 24 h, and the temperature was maintained at 37 ± 0.5° C. Once the equilibrium was attained, the resulting mixture was centrifuged  at 8° C for 1 h at 15,000 rpm. The formed supernatant was then diluted with a suitable quantity of methanol and analyzed quantitatively for ITT concentration using a UV-Visible spectrophotometer at 362 nm. The experiment was repeated three times, and the values were noted as mean ± standard deviation.2.3.Nine nanoemulsion formulations were designed with different proportions of the selected oils, surfactant, and co-surfactant using different concentrations of ITT (20–60 mg) in the first formulation only, then ITT concentrations from 20 to 40 were used in formulations 2 and 3. After that, the drug concentration that yielded the lowest globule size in formulations from 1 to 3 was used in the rest of the formulations. The composition of the 9 formulations was presented in The SNEDDs were prepared as follows; ITT was added to the oil phase mixture; subsequently, a mixture of surfactant and co-surfactant was added. The formed mixture was further sonicated using Ultrasonic Processors . Finally, the formed dispersion was added to distilled water drop by drop, and nanoemulsion was formed spontaneously on mixing at room temperatures  , surfactant [labrasol] (X2), and cosurfactant [transcutol] , steady-state inflow (JSS) (Y2), and penetrability percentage (Y3) was studied  has proven a proficient tool in optimizing the various parameters in a multifarious process to obtain the desired responses; thus, it has been applied to formulate ITT-SNEDDS. Among several models, the mixture design was selected to study the interaction of independent variables. Three factors were selected, namely, percentages of oil mixture [tea tree oil/baobab oil in ratio 1:1]  with multiple response optimization and RSM. Optimized formulation was selected based on the constraints of selected responses.2.8.After the optimization of experimental runs, an optimized formulation was prepared according to the concentrations proposed by the software, and its various parameters were evaluated. The rationale for the selected experimental design was validated by comparing the experimental values with predicted values and calculating relative error (%). The following equation was used to calculate relative error  was utilized. First, the dialysis membrane was soaked in phosphate buffer (pH 7.4) for 15 min at 25 ± 0.5° C. The receptor compartment was filled with phosphate buffer, and glass tubes covered with the dialysis membranes and filled with certain volume of the optimized formulation equivalent to 5 mg of each drug were placed in the medium with continuous rotations (75 rpm). The temperature was maintained at 37 ± 0.5° C throughout the experiment. Samples were withdrawn at various time intervals , and ITT and RSV concentrations were analyzed at 362 and 299 nm, respectively.In vitro, drug release studies were performed to calculate the amount of ITT and RSV released for the optimized formulation according to a previously described method . Low hea2.10.The permeation study for optimum formulation was carried out as previously described in Section 2.6.2, however the permeation parameters for both ITT and RSV were calculated.2.11.Thirty-six male adult mice weighing 20.5 ± 1.5g were selected and adapted for seven days before the study. The animals were divided into six groups consisting of six animals in each group. Animal groups received the following treatments; group 1 received normal saline orally and served as negative control, while group 2 received oral aqueous dispersion of ITT. Moreover, group 3 received topical aqueous dispersion of ITT, group 4 received topical optimized ITT-SNEDDS formulation, group 5 received optimized topical ITT-RSV-SNEDDS and finally group 6 received the marketed topical cream. The dose of ITT in tested formulations was 5 mg/kg, and the treatment period was 14 days. The tested rats were maintained at temperature of 25 ± 0.5 °C and 30 − 70% of relative humidity and subjected to 12 h darkness and 12 h light cycle. At the end of the study period, animals were sacrificed with cervical dislocation, blood samples were instantly withdrawn and finally dysfunction of the liver was estimated by measuring AST, ALT, superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px).2.12.in vivo results obtained were expressed as mean ± S.D and analyzed using Statistical Package for the Social Science, version 26 . One-way ANOVA was followed by a post-hoc Tukey test to determine significant differences between quantitative variables. p Value <.05 was noted as statistically significant.The 3.3.1.The solubility of ITT in assorted vehicles is depicted in Also, ITT exhibited highest solubility with transcutol co-emulsifier (24.34 ± 14.28 µg/ml). Transcutol is known for its great miscibility in polar and non-polar solvents, as well as its competence to increase the absorption and degree of permeability , maximum JSS (Y2), and high permeability percent (Y3)  and the sequential sum of squares (Type-I), different models were selected , the model reduction could have been required. The Lack of Fit F-value (0.97) was non-significant, with only a 1.25% chance that the Lack of Fit F-value this large could occur due to noise.The selected model was statistically significant, F = 41.64, Furthermore, Adeq Precision measures the signal to noise ratio. In general, a ratio greater than 4 is desirable. The obtained ratio (17.910) indicated an adequate signal to navigate the design space  was affected significantly by the synergistic effect of liner mixture (p < .0001)  = 481.52 ×1 + 110.81×2 + 260.11×3Globule size  and Lack of Fit F-value (0.7014) for Response (Y2) confirmed that the selected model is significant and the lack of fit is not significant, relative to the pure error. Adeq Precision (17.910) indicated an adequate signal to navigate the design space. Based on ANOVA results  factors affected Response II antagonistically at a p value <.0001, and all remaining factors showed a synergistic effect. The generated polynomial equation shows that all these selected variables significantly affected JSS. Mixture design contour plots and 3D plots were similar to those for Y1. As discussed in the previous sections, higher concentrations of surfactants and oil mixture account for elevated and decreased JSS, respectively.The model F value  = − 12298.76 ×1 + 3087.44 ×2 − 494.73 ×3 + 17529.79 X1X + 25207.50 X1X3 − 4205.41 X2X3 − 21726.95 X1X2X3 +17752.15 X1X2 (X1−X2) + 13269.82 X1X3 (X1−X3) − 5101.24 X2X3 (X2−X3)J3)% permeated . The selected three components are depicted in the corners of the triangle, and 3 D contour plots were drawn to study interaction effects. The fitted linear special regression equation was generated, as shown below.Adequate Precision for the linear model of YFinal Equations with Coded Factors1 + 69.94 ×2 + +45.46 ×3% Permeated = 5.02 ×The above results showed a higher significant effect of surfactant concentration compared to other variables. As the higher surfactant proportions can reduce the globule size, this can enhance the permeation percentage  and 62.5% for globule size, Jss and permeation percent, respectively.The process was optimized by setting the goals for every response and simultaneously applying the Global desirability function (D). Based on these criteria, the desirability plot was generated with a D value of 0.918 . In conc3.4.SS, and percentage permeation. Relative error was calculated by comparing the predicted and practical values, as shown in In the end, the experimental design was further validated to confirm accuracy. Optimized formulation was prepared with the given conditions and evaluated for globule size, J3.5.SS values further supported this finding. ITT had more JSS values, resulting in higher permeation. In general, RSV has a slow permeation, but it can exhibit and retain antioxidant and anti-inflammatory effects on the skin.The permeability percentage through cellulose membrane for RSV and ITT was found to be 42.52 ± 2.02% and 58.66 ± 0.77% after 12 h . JSS val3.6.Ex vivo studies were conducted using simple, low head dissolution apparatus to establish the effect of ITT-RSV formulation. The permeation was studied using rat full-thickness skin, and the results are summarized in ex vivo permeation diagram of ITT and RSV from optimized RVS-ITT-SNEDDS formulation.3.7.Despite several notable advancements, hepatic safety and hepatic cell regeneration products are inadequate for treating several liver disorders. Numerous herbal extract formulations were also used in this approach  emerged between RSV-ITT SNEDDS formulation and the control formula for all tests. To conclude, the advanced formulation was developed relative to the marketed product in terms of hepatoprotective efficacy.The pretreatment with a formulation containing ITT + RSV for 14 days significantly increased the antioxidant property and reduced the lipid peroxidation, which reduced overall oxidative stress. All changes obtained with RSV formulation (Group 5) were more beneficial in ameliorating hepatotoxicity compared to other groups. Additionally, these two groups differed significantly from a marketed product (Group 6). No significant difference  were selected to formulate the nanoemulsion. A low concentration of ITT favored the formation of globules with smaller particle sizes. Mixture design was successfully applied to optimize the concentrations of various parameters according to the desirability approach. Formulation with 0.15 g of oil mixture, 0.6 g of surfactant (labrasol), and 0.250 g of co-surfactant (Transcutol) achieved the optimum formulation. The range of relative error was acceptable (<5%), confirming the preciseness of the design. Click here for additional data file.


P < 0.01). Breast muscle percentage was significantly decreased at 0.02% quercetin (P < 0.01), and thigh muscle percentage tended to increase (P = 0.078). Meanwhile, 0.04% and 0.06% quercetin significantly decreased TG (P < 0.01), TC (P < 0.01), and LDL content (P < 0.05) in serum. Serum leptin and adiponectin contents were significantly increased by 0.04% and 0.06% dietary quercetin supplementation, compared with the control (P < 0.01). Analyses of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database were used to identify differently expressed genes and lipid metabolism pathways. Quercetin decreased abdominal fat percentage through regulating fat digestion and absorption, glycerophospholipid metabolism, AMPK signaling pathway, fatty acid degradation, and cholesterol metabolism.This study is aimed at evaluating the regulatory mechanism of quercetin on lipid metabolism in the ileum of broilers to better understand these pathways decreasing abdominal fat. 480 chickens were randomly divided into 4 groups . Breast muscle, thigh muscle, and abdominal fat pad were removed and weighed at 42 d of age. Serum was obtained by centrifuging blood samples from the jugular vein (10 ml) to determine high-density lipoprotein (HDL), total cholesterol (TC), low-density lipoprotein (LDL), triglyceride (TG), leptin, and adiponectin using ELISA. About 5 g of the ileum was harvested and immediately frozen in liquid nitrogen for RNA-seq. Then, the confirmation of RNA-seq results by the Real-Time Quantitative PCR (RT-qPCR) method was evaluated using Pearson's correlation. Compared with control, abdominal fat percentage was significantly decreased with increasing quercetin supplementation, and the best result was obtained at 0.06% dietary quercetin supplementation ( Meat is an important protein source, and chicken is one of the most popular food commodities in the world and the second most preferred meat for Chinese consumers. The fat of meat is an important component in meat quality and impacts animal productivity. The breeding of poultry focused on increasing growth performance and improving breast and thigh meat yields over the past decades. Growth performance made great progress; however, there has been excessive deposition of abdominal fat. In order to reduce fat deposition and improve feed efficiency, different feed additives have been adopted in broiler production. As a kind of safe feed additives, quercetin is impressive .Quercetin is a flavonoid abundant in onions, apples, tea, and red wine, which exhibits antiviral, antitumorigenic, and anti-inflammatory effects –5. MoreoThe intestine plays a vital role in fat digestion and absorption; both the jejunum and ileum were involved in the absorption of fatty acid (FA) in laying hens and broilers , 13. EstThe transcriptome is a necessary link between genomic/genetic information and the biological functions of the proteome. Of these, RNA-seq has been widely utilized to detect differentially expressed genes (DEGs) between two gene expression patterns and causative variants. Many studies of RNA-seq have been conducted in the intestinal mucosa , heart , uterineThe objective of this study was to evaluate the regulatory mechanism of quercetin on carcass characteristics in broilers. The results of this experiment will provide scientific basis for quercetin application in animal production.Arbor Acre broilers  from a commercial company  were purchased and randomly divided into 4 groups with 6 replicates per group and 20 broilers per replicate. The broilers were housed in wire cages . Lighting (16 h light : 8 h darkness) was provided during the 6-week experimental period, and temperature was maintained at 32 to 34°C in the starting 3 days and decreased by 2 to 3°C per week to a final temperature of 24°C. Humidity of the experimental room varied from 60% to 65%. Experimental diets and water were available ad libitum. The composition of the basal diets is shown in n = 12 per group). After 12 h fasting, blood samples from the jugular vein (10 ml) were obtained from each group and kept in ice. Serum was obtained after centrifugation at 4°C, 3000 × g for 15 min, and stored at -20°C until analysis. Breast muscle, thigh muscle, and abdominal fat pad  from one bird of average BW per replicate were removed and weighed at 42 d of age. To compensate for the differences in carcass weight, these values were expressed as a percentage of carcass weight. About 5 g of ileal mucosa was harvested and immediately frozen in liquid nitrogen, and kept at -80°C for further RNA isolation. All procedures used in this study were approved by the Animal Care and Use Committee of the University. Housing, management, and care of the birds confirmed to the guidelines of the Agricultural Animal in Agricultural Research and Teaching of Heilongjiang Province .At the end of experiment (42 d), two broilers per replicate were selected (Content of high-density lipoprotein (HDL), LDL, TG, TC, leptin, and adiponectin were determined using an enzyme-labeled instrument according to ELISA kit instructions .In this endeavor, twelve samples of ileal mucosa were sequenced using the HiSeq 2000 System  by Genesis (Beijing) Co. Ltd. The data analysis included sequencing data filtering, read mapping, transcript and gene identification, analysis of differential gene expression, and functional annotation.μl of reaction mixture containing 5 μl of total RNA, 4 μl of 5 × Prime Script Buffer, 1 μl of PrimeScript RT Enzyme Mix I, 1 μl of Oligo(dT) Primer (50 μmol/l), 1 μl of Random Primers 6 mers (10 μM moI/l), and 8 μl of RNase Free dH2O  was incubated in the following conditions: reverse transcription at 37°C for 15 min and inactivation of reverse transcriptase at 85°C for 5 min, until temperature was decreased to 4°C. Successful cDNA synthesis was confirmed by amplifying the β-actin amplicon using PCR. cDNA was amplified using a 20 μl PCR reaction system containing 2 μl of cDNA, 10 μl of 2 × SYBR Green PCR Mix, 0.4 μl of 50 × ROX Reference Dye II , 0.8 μl of PCR Forward Primer, 0.8 μl of PCR Reverse Primer , and 6 μl of ddH2O. The following PCR conditions were used: initial denaturation at 95°C for 30 s, followed by PCR reaction at 40 cycles of 95°C for 5 s and 60°C for 34 s, and melting curve analysis at 95°C for 15 s, 60°C for 2 s, and 95°C for 15 s. The PCR products were verified by electrophoresis on 1% agarose gel and DNA sequencing. Standard curves were generated using pooled cDNA from the assayed samples, and the comparative cycle threshold method (2−ΔΔCT) was used for quantifying mRNA levels.To confirm differential expression of genes, real-time quantitative polymerase chain reaction (RT-qPCR) assays for 12 randomly selected DEGs in the same RNA samples were determined using RNA-seq. All gene expression levels were measured using RT-qPCR . The primer sequences for broilers  were desP<0.05 were accepted as statistically significant, and all the results were expressed as the “mean values ± standard deviation.”The data from this experiment were subjected to one-way ANOVA as a completely randomized design with 4 treatments and 6 replicates in each treatment. The data were submitted to ANOVA, using SPSS 20.0 software . Calculated△Ct (corrected sample) = the mean value of the target gene − the mean value of the internal reference gene; △△Ct = △Ct − the mean value of the control group. Differences among treatment means with a probability level of P = 0.078); however, abdominal fat percentage was significantly decreased with increasing quercetin (P < 0.01) (P < 0.01) compared with the control.Thigh muscle percentage tended to increase ( < 0.01) , and breP > 0.05); however, 0.04% and 0.06% quercetin significantly decreased the content of TG (P < 0.01), TC (P < 0.01), and LDL (P < 0.05) in serum. Contents of serum leptin and adiponectin were significantly increased by 0.04% and 0.06% dietary quercetin supplementation (P < 0.01)  .Gallus gallus, 5.0). Sequence data from the 12 samples were mapped to the reference genome  compared with the control using GO enrichment and KEGG pathway analyses to understand the regulatory network of lipid metabolism. GO is a type of biological ontology language, which is divided into three parts, including biological process, cellular component, and molecular function. Unlike the functional annotation of a single gene, a gene functional enrichment analysis is based on GO entries, and the results may directly reveal the overall functional characteristics of an entire list of genes. Metabolic processes accounted for the second in the most significantly enriched GO term .The results showed that 9039 DEGs of control/0.02% quercetin were annotated into 336 pathways, 11842 DEGs of control/0.04% quercetin were annotated into 337 pathways, and 9970 DEGs of control/0.06% quercetin were annotated into 337 pathways, including three different classifications . The KEGP < 0.01), AMPK signaling pathway (P < 0.01), cholesterol metabolism (P < 0.01), steroid hormone biosynthesis (P < 0.01), insulin resistance (P < 0.05), bile secretion (P < 0.01), and metabolic pathway (P < 0.05) in 0.02% quercetin. There were differential gene enrichment pathways including cholesterol metabolism (P < 0.01), insulin resistance (P < 0.05), and fatty acid metabolism (P < 0.05) in 0.04% quercetin. And there were differential gene enrichment pathways including fat digestion and absorption (P < 0.01), glycerophospholipid metabolism (P < 0.01), fatty acid degradation (P < 0.05), metabolic pathways (P < 0.05), and ether lipid metabolism (P < 0.05) in 0.06% quercetin , affirming the findings of Ma et al. [Ginkgo biloba leaves (including abundant flavonoids) in broilers decreased abdominal fat deposition [P < 0.05) in broilers. The current results are supported by previous studies on the effect of flavonoids on chicken quality in broilers [Flavonoids belong to a group of natural substances, which have a variable phenolic structure and are abundant in fruit, vegetables, tea, and wine. Quercetin is one of the most abundant flavonoids . Differea et al. . Too muca et al. . Diet suposition . Moreoveposition . Our resbroilers , 28.Chrysanthemum indicum ethyl acetate (CIEA) significantly decreased serum lipid profiles, including TG and LDL [P < 0.01), and LDL (P < 0.05) in broilers. This further confirmed the hypolipidemic effects of quercetin, just like the other flavonoids.Lipids mainly include triglyceride (TG), phospholipids, and cholesterol (CHO), and the content of TG and CHO are key indicators of lipid metabolism. Hesperidin and naringin mainly regulated lipid metabolism by reducing the content of TG and CHO in the human liver . Further and LDL . The curM. citrifolia leaf extracts (MLE60) compared to control (535.3 pg/ml) [Poncritus trifoliate leaf extracts which increased adiponectin levels in serum of HFD-fed mice [P < 0.01). Meanwhile, together with the results for TG in our experiment, our findings were supported by Nepali et al. and Ma et al. [Leptin is a hormone synthesized by white adipose tissue (WAT) and plays an important role in weight control by suppressing food intake and increasing energy expenditure. Leptin is also a regulator of cellular TG content . Adipone3 pg/ml) . The resfed mice . The prea et al. , 29, whoRecently, the RNA-seq technique has been a powerful and revolutionary approach to quantify gene expression levels and survey detailed transcriptome profiles at unprecedented resolution and sensitivity , 39. In 2FC, the upregulated genes are mainly related to amino acid metabolism , while the downregulated genes are mainly related to lipid metabolism . IDH1 is involved in the tricarboxylate cycle and plays a role in energy metabolism and biosynthesis in organisms. PSAP is a lysosomal gene which delivers bound sphingolipids to cell plasma membranes and enters an endocytotic pathway, responsible for digesting the lipoproteins [In the present study, among the top 10 of the significantly upregulated and downregulated genes between quercetin  and the control group according to the logproteins –41. CD36proteins .After functional enrichment analyses, GO terms and KEGG pathways were mainly involved in cellular and metabolism processes . The resP < 0.01). APO A1, MTTP, ABCG5/8, and PLA2G12B are important genes involved in fat digestion and absorption, which are highly expressed in 0.02%, 0.04%, and 0.06% quercetin treatments. Secreted PLA2G12B is a novel mediator of TG metabolism [2FC of 2.01, 2.16, and 1.61, respectively. Furthermore, another significantly differential gene, APO A1, belongs to an APO family that encodes important regulators of lipid biosynthesis and metabolism [2FC records, the expression of APO A1 was increased by 2.1-, 1.37-, and 1.95-fold as much as the control for 0.02%, 0.04%, and 0.06% quercetin. Meanwhile, together with the results for HDL in this experiment, our findings were supported by Zhou et al. [There were 115 DEGs in this pathway, and 0.06% quercetin was significantly different from the control . There are four isoforms for LPCAT  [PLD1 and LPCAT3 are the two main genes that are associated with the glycerophospholipid metabolism. There were 105 DEGs in this pathway, and 0.02% quercetin and 0.06% quercetin were significantly different from control ( LPCAT4) . Plasma  LPCAT4) . LPCAT3  LPCAT4) . Again,  LPCAT4) . PLD hyd LPCAT4) . PLD1 kn LPCAT4) . PLD1 kn LPCAT4) . Further LPCAT4) . The aboP < 0.01). The CD36, PRKAB2, and PI3KR1 genes involved in the AMPK signaling pathway were differentially expressed compared with the control. CD36 is a membrane receptor that facilitates long-chain fatty acid uptake and plays an important role in mitochondrial oxidation [There were 505 DEGs in this pathway, and 0.02% quercetin was significantly different from control . ACSLs , ADH1C, and CPT1A were upregulated by dietary quercetin supplementation. ACSLs are essential for de novo lipid synthesis, fatty acid catabolism, and remodeling of membranes [β-oxidation [P < 0.05). ADH1C  is the predominant isozyme expressed in duodenal, jejunal, and ileum mucosa [γ1 and γ2). It also directly interacted with the high-density lipoprotein (HDL) [P < 0.01), while ACSLs and ADH1C directly interacted with TG and HDL, respectively, together with the results on TG and HDL in this experiment, thereby decreasing abdominal fat percentage.In the present study, compared with the control, 0.06% quercetin was significantly different from the control in the fatty acid degradation pathway  . The preP < 0.01). APO A4, APO B, and APO C3 also belong to an APO family that encodes important regulators of lipid biosynthesis and metabolism [P < 0.05). APO B located on the surface of the lipoprotein particles is the main lipoprotein of low-density lipoprotein taking charge of transferring cholesterol into tissues. Total flavonoids extracted from Polygonum perfoliatum L. (TFP) decreased the level of APO B and increased the level of APO A, thus adjusting the ratio of APO A/APO B and the metabolic disturbance of lipoprotein by TFP treatment in hyperlipidemia rats [APO A4, APO B, APO C3, and SCARB1 are key transcription factors in the regulation of cholesterol metabolism. There were 217 DEGs in this pathway, and 0.02% quercetin and 0.04% quercetin were significantly different from the control (tabolism . APO A4 tabolism . Additiotabolism . The resmia rats . Howevermia rats . APO C3 mia rats . Meanwhimia rats . Our resIn summary, our results showed that leptin and adiponectin directly interacted with TG, thus decreasing abdominal fat percentage. Meanwhile, quercetin decreased TG, TC, and LDL via regulating APO A4, APO B, APO C3, APO A1, MTTP, ABCG5/8, PLA2G12B, CD36, PRKAB2, PIK3R1, ACSLs, ADH1C, LPCAT3, PLD1, and SCARB1 gene expression in ileal mucosa of broilers.Quercetin decreased abdominal fat percentage through regulating the signaling pathway, including fat digestion and absorption, glycerophospholipid metabolism, the AMPK signaling pathway, fatty acid degradation, and cholesterol metabolism.


Diaporthe eres has been recently reported as the causal agent of hazelnut defects, with characteristic brown spots on the kernels surface and internal fruit discoloration. Knowledge regarding the ecology of this fungus is poor but, is critical to support a rationale and effective hazelnut crop protection strategy. Therefore, a study was performed to describe and model the effect of different abiotic factors such as temperature  and water activity  regimes on D. eres mycelial growth, pycnidial conidiomata development and asexual spore production during a 60-day incubation period. Alpha conidia germination was tested in the same T range and at different relative humidities  over 48 h incubation period. Fungal growth was observed from the first visual observation; regarding pycnidia and cirrhi, their development started after 8 and 19 days of incubation, respectively and increased over time. The optimum T for growth was 20–25°C and for pycnidia and cirrhi development was 30°C; aw ≥ 0.98 was optimal for the tested steps of the fungal cycle. The best condition for conidial germination of D. eres was at 25°C with RH = 100%. Quantitative data obtained were fitted using non- linear regression functions , which provided a very good fit of the biological process (R2 = 0.793–0.987). These functions could be the basis for the development of a predictive model for the infection of D. eres of hazelnuts. Diaporthe eres was recently reported as the causal agent of hazelnut defects, which produces brown spots on the kernel surface or internal discoloration, which become visible inside the fruits after being cut in half . D. lifornia , associalifornia  and withlifornia . Moreoveavellana , and it avellana , peach [avellana , pear [9avellana , and graavellana , 11–13.Diaporthe is the name recently recommended for use by the scientific community instead of Phomopsis, the asexual stage, more frequently observed [Phomopsis/ Diaporthe spp. have been associated with several important diseases, with quite different symptoms, described both in annual and perennial plants [observed . Phomopsl plants , 15.Diaporthe spp. are considered monocyclic pathogens [D. eres [D. eres is very scarce, although some knowledge is available in other crops like Vitis vinifera [Prunus persica [Rubus sp. [Juglans cinerea [Diaporthe/Phomopsis on different crops have only sporadically been considered, except for P. viticola and D. helianthi [Based on the literature, athogens . Thus, tathogens . Tempera[D. eres . Howevervinifera , Prunus  persica , Rubus subus sp.  and Jugl cinerea  and fore cinerea ; furtherelianthi , 22.w), pH, atmosphere composition, substrate, interaction among co-occurring microorganisms and time [w are considered to be the most critical abiotic factors that define the ability of fungi to grow on plants and fruits [w. In fact, fungi could growth at temperature below 0°C up to above 40°C, while the minimum aw at which growth of fungi has been observed is about 0.61. The minimal aw values for sporulation of fungi have been investigated for only few species, but data suggested that higher aw is required for spore formation than for their germination [The fungal growth, conidia sporulation and germination are influenced by several variables, such as temperature, water activity  and Water Agar  were used to perform this study. The water activity (aw) of PDA and WA was aw = 0.99; glycerol was added to the media, according to Dallyn and Fox (20), to have aw = 0.83, 0.87, 0.90, 0.93, 0.96 and 0.98 for PDA and aw = 0.94 and 0.97 for WA. The accuracy of the aw modifications (±0.005) was confirmed using an Aqualab LITE .Diaporthe eres (PH01), isolated from defective hazelnut kernels and confirmed by morphological and molecular methods as described in Battilani et al. [A strain of  eres PH0, isolateD. eres PH01 was grown on WA plates and incubated at 25°C for one week with 12-hour light photoperiod to be used as an inoculum for the planned trials. Sterilized transfer tubes, 5mm Ø, were used to take plugs from the margin of the one-week WA incubated plates and transferred to the center of unmodified PDA agar media  or to those modified aw conditions.w regimes. All the studies were carried out twice.The study considered fungal growth, conidiomata development and maturation, α conidia production and their germination in different T and aD. eres growth, PDA Petri dishes (aw = 0.99) were centrally inoculated with D. eres WA plugs, prepared as previously described. The dishes were incubated at different T regimes, from 5 to 35°C (steps of 5°C).To study the impact of temperature (T) on w regimes, PDA plates with modified aw (0.83–0.99) were centrally inoculated with D. eres WA plugs and incubated at 25°C. All the replicates and treatments were incubated in the dark for 60 days.Similarly, to study the impact of ath day; furthermore, additional measurements were made after 45 and 60 days in the studies of the effect of T regimes. The studies were carried out with 5 replicates per treatment.In each replicate plate, two perpendicular colony diameters were measured every 3–4 days up to the 30The Petri plates used for the growth study were also observed for the occurrence of pycnidial conidiomata and the presence of cirrhi with the same time frames detailed previously.D. eres culture plates, for each tested T, after 60 days incubation and transferred into 1.5 ml Eppendorf tube® with 200 μl of double-distilled water, crushed and made up to 1 ml with double-distilled sterile water. The number of α conidia was then counted using a Bürker chamber.To quantify the conidial production, 5 pycnidial conidiomata with visible cirrhi were randomly selected from D. eres colonies incubated at 25°C for 30 days. The concentration of the conidial suspension was adjusted to 105 α conidia/ml using a Bürker chamber.Conidia were collected from cirrhi produced by pycnidial conidiomata of ® and incubated from 5 to 45°C, with 5°C steps, for 48 h. The experiment was carried out with 5 replicates per treatment.Ten μl of the conidial suspension were used to inoculate 5 mm Ø WA plugs, placed on a microscope glass slide; the glass slides were incubated inside 90 mm Ø Petri dishes with wet filter paper on the bottom, according to Ciliberti et al. . The filReplicates were destructively sampled after 6, 12, 24 and 48 h. The WA plugs were stained with lactophenol blue and observed using an optical microscope . Spores were considered germinated when the germ tube was visible. Fifty spores were observed for each combination of T × RH and incubation time, for each WA plug; spore germination was calculated as follows:w regimes, were considered separately for each incubation time. They were standardized , to obtain growth rates on a 0–1 scale, with 0 = no growth, and 1 = maximum growth. Growth rates of D. eres, at all incubation times, were then jointly analyzed. The same approach was applied to obtain the % of α conidial germination on a scale of 1–100, at different T or RH regimes.Statistical analyses were performed using IBM SPSS Statistics v.25 . Data on fungal growth, based on the fungal culture diameter, at different T or aw, RH and GDD). The equation parameters were estimated applying the non-linear regression procedure of the statistical package PASW IBM SPSS Statistics v.25  which minimizes the residual sum squares using the Levenberg-Marquardt algorithm. The best model was chosen based on the adjusted R2 and on the minimum number of iterations required by the algorithm to converge on parameter estimates, as indicators of goodness of fit. Minitab 18  was used to develop the surface response contour plots of α conidial germination data, on the combinations of T × RH. For each combination, the germination rate, computed as previously described, was used as the input for data plotting.For pycnidial conidiomata production, they were reported based on Growing Degree Days (GDD); -the GDD was calculated as the summation of incubation T over time. The number of pycnidia counted was standardized on the maximum value observed and rated on a 0–1 scale. One-way-ANOVA for repeated measure using the Greenhouse-Geisser correction factor was applied to fungal growth , α conidial germination , pycnidial conidiomata and cirrhi occurrence  and α conidial production by pycnidial conidiomata (n. α conidia/pycnidium). The Tukey test was applied to highlight significant differences between means. Different non-linear regression models were fitted to the rate data to describe fungal growth, pycnidial production and α conidial germination as function of the ecological factors .Fungal growth was observed at all the tested Ts, between 5 and 35°C, and measured as colony diameter. Temperature regimes significantly influenced fungal growth (p≤0.001); the maximum diameter was measured at 20–25°C (mean 87.1 mm) and the minimum at 35°C ; the optimal growth condition was at aw = 0.99 (colony diameter = 85.3 mm), and this was decreased significantly at each aw change, until aw≥0.90 . The maximum diameter measured at the end of incubation period for all the aw treatments are presented in https://doi.org/10.6084/m9.figshare.13168532).Pycnidial conidiomata were observed at all the tested Ts, except at 35°C; cirrhi were detected only at 25°C and 30°C.https://doi.org/10.6084/m9.figshare.13168532).The ANOVA showed that T significantly affected (p≤0.001) pycnidial conidiomata production. Optimal T for the pycnidial conidiomata occurrence was observed at 30°C, with a mean number of pycnidial conidiomata of 38.4 in each dish. The occurrence of pycnidial conidiomata was initiated after 8 days incubation (mean = 0.6 pycnidia/colony) and increased up to the end of the trial . Furthermore, the highest occurrence of pycnidia with cirrhi, (30%) was observed at 30°C, and remained unchanged from 45 to 60 days incubation (the raw data are reported in https://doi.org/10.6084/m9.figshare.13168532).The occurrence of cirrhi was detected after 19 days of incubation (mean number: 0.3), increasing up to the 60The concentration of α conidia in cirrhi was assessed after 60 days incubation. At the extreme Ts tested (5 and 35°C), no conidia were found.w≥0.96 and cirrhi at aw≥0.98, while they did not develop at lower aw levels. The ANOVA showed that aw was a highly significant (p≤0.001) factor for pycnidial conidiomata occurrence. The optimum aw for pycnidial conidiomata development was with freely available water (aw = 0.99), with the mean n. pycnidial conidiomata/colony = 60.1. Their occurrence started after 15 days incubation  and increased up to the end of the study .The pycnidial conidiomata were observed at aw (p≤0.005). The optimum aw for cirrhi production by pycnidia was recorded at aw = 0.98, with 8% of pycnidia with cirrhi after 30 days incubation. The occurrence of cirrhi was initiated after 26 days incubation  and increased up to the end of the study .Regarding pycnidia showing cirrhi, they were significantly affected by ahttps://doi.org/10.6084/m9.figshare.13168532).Germination of α conidia was significantly affected by both T and RH (p≤0.001). The optimum T for α conidial germination was at 25°C, with a germination rate of 42.5%. Regarding RH, the highest α conidial germination mean rate (29.1%) was at RH = 100% and decreased with RH decrease. After 6h incubation, the mean percentage of α conidia germinated was 4.4%, and germination rate increased with time, until around 32% after 48h. No germination was observed at 5 and 45°C = both for T  and aw , with sw influences quality parameters, including mold if moisture is too high, shrivel if too low, color changes, and rancidity [w is the deterministic factor for fungal growth and was reported that field fungi require aw>0.90 to survive [w measured on hazelnut changes over crop growing period with values range between 0.72 and 0.99, with risk of fungal growth in field.It is well known that many extrinsic factors, including humidity and temperature, can affect the quality of hazelnuts. The moisture represents one of the most important factors, since aancidity . The stuancidity  conclude survive –36. The D. eres to grow and develop reproductive structures over a broad range of Ts (5–35°C) and aw levels (0.83–0.99) were considered in this study for the first time. D. eres colonies grew to around 28 mm in diameter in 5 days on PDA, when the mean growth over the range of Ts was considered. No data on this fungal species are available in literature considering the same range of Ts discussed in this work. Previous studies on Diaporthe/Phomopsis spp. reported growth of 5–20 mm for D. tanakae and a growth > 70 mm for P. mali and P. perniciosa [D. eres colonization rates in the same time frame and T conditions was 63 mm. This wide variability in relative growth between Diaporthe species was also found by Guarnaccia et al. [D. bohemiae, D. celeris, D. hispaniae and D. hungariae had variable capacity for colonization of defined media between 6 and 15 days at 21°C. However, no studies have reported intraspecific variability.The capacity of rniciosa  on PDA ma et al. . They foDiaporthe/Phomopsis spp. was observed in a T range of 18–30°C, depending on the species. Thus, optimal T ranges between 23–30°C was reported for Phomopsis sp. isolated from cashew leaves [D. ueckerae, the causal agent of dieback disease on Michelia shiluensis [P. eucommicola, cause of canker disease in poplar [P. vaccini was between 20–28°C [P. amygdali [P. cinerascens [P. asparagi [Phomopsis sp isolated from Eucommia ulmoides [P. destruens [D. eres as the causal agent of shoot blight of peach trees in Greece reported an optimum T for mycelial growth on PDA at 25°C, although no growth was observed at 35 and 10°C after 5-day incubation periods [The maximum growth of w leaves , 25–30°Ciluensis  and betwn poplar . However 20–28°C , at 25°Camygdali , P. cineerascens  and P. aasparagi , 28°C foulmoides  and 20°Cestruens . A studyw≥0.90 was the best for D. eres growth, with aw≥0.96 and aw≥0.98 for pycnidia and cirrhi development, respectively. Previously, RH values > 95% was claimed to be necessary for P. amygdali mycelial growth, pycnidial conidiomata development, asexual spore production and their germination [In the present study, amination .2 of colony recorded at 30°C; pycnidia were not detected at 35°C, as also observed for D. ueckerae by Yi et al. [Phomopsis sp. isolated from raspberry canes and P. cinerascens between 15 and 25°C [In this study, the number of pycnidial conidiomata counted in the fungal colonies varied at different incubation Ts, with an optimum of 0.9 pycnidia/cmi et al. . Other sand 25°C , 47.D. eres was observed at 30°C in this study, with 6.3 compared to 0.2 at 25°C; no cirrhi were observed at all the other Ts and incubation times examined. In addition, aw influenced cirrhi occurrence with production only observed at aw≥0.98. The time required for pycnidial conidiomata to produce cirrhi was around 4–5 weeks, according to Rosenberger [D. perniciosa and P. tanakae. The lower and upper T limits for cirrhi development reported for P. viticola are respectively 4 and 36°C, with the optimal T at 22°C. Anco et al. [w on cirrhi production by Diapothe/Phomopsis spp.The maximum number of pycnidia with cirrhi/colony and cirrhi production by enberger  in D. peo et al.  observedDiaporthe spp are considered monocyclic [P. tanakae developed pycnidia in May and cirrhi were observed in late May; conidia were released from the fruiting structures only during rainfall between June and September [P. viticola [The time necessary for pycnidial conidiomata and cirrhi development is quite long; indeed, diseases caused by nocyclic , 49. In eptember . The funeptember , as alsoviticola .Phomopsis/Diaporthe produce two types of conidia: elliptical and biguttulate α conidia and long and thin β conidia; the latter were for an extended period characterized as being unable to germinate [erminate , 51. Morerminate ; however5 α conidia/pycnidium). In fact, according to Nabetani et al. [P. vaccini contain 1–2×104 conidia/pycnidium, which was considered to be a high concentration of conidia with a crucial role in disease epidemics. Moisture and recurrent precipitation events were found to promote conidial dispersal and germination, resulting in infections of the twigs, blossom blight and stem dieback of highbush blueberry.Concentration of α conidia in cirrhi was scarcely influenced by T. However, they were strongly affected by the incubation time: in the range of Ts considered in this study, the α conidial concentration was abundant, and probably sufficiently high as a source of inoculum for the pathogen infection in the field . It was therefore confirmed the germination only over a very narrow range of RH, with a wide range of T.The capacity of D. viticola α conidia were reported to germinate in a T range of 1–37°C, with an optimum of 23°C in a few hours in free water or near 100% RH [D. viticola α conidia germinated in 4 h at 25°C, while, at least 7–10 h with high RH conditions were necessary with 15–18°C [Previously,  100% RH , with st 15–18°C .w on D. eres growth were modelled using a Bete and a logistic equation, respectively. The approach has been widely described in the literature for other pathosystems, e.g., powdery mildew on cucumber [Guignardia bidwellii conidia from overwintered grape berry mummies [Aspergillus carbonarius spores on grapes and synthetic medium [Fusarium graminearum in wheat [F. langsethiae on synthetic medium [The effects of T and acucumber  and maizcucumber , as wellcucumber . The procucumber , recentl mummies . The fit mummies –60 makesc medium , the gerin wheat  and the c medium .D. eres, they can be assumed as representative, because intraspecies variability was never reported. There are several examples regarding the projection of quantitative data on fungal ecology in predictive models, like Botrytis cinerea for grapes [Fusicladium eriobotryae in loquat [D. eres in hazelnut.All the data fitting was successful, with very good statistical evaluation; therefore, some bricks for the future building, a mechanistic predictive model, are available and contour of suitable conditions defined. Even if based on studies managed with only one strain of r grapes  or Fusicn loquat , and thaD. eres. As D. eres is a monocyclic pathogen [However, it is important to underline that there is still a lack of knowledge on the specific pathosystem for hazelnut-pathogen , it willpathogen . Fill thS1 Table(XLSX)Click here for additional data file.


In addition, the P value for the 1-mo CD150lo time point should have been **, P < 0.01, not ****, P < 0.0001. The corrected figure and legend are shown here. The authors also clarify that the P values in Fig. 2, E–G; Fig. 5, A–C and F–I; Fig. 6, F–J; Fig. 7, D–H; Fig. 8; and Fig. 9, A–C, E–K, and M–P depict results derived from the unpaired Student’s t test. The one-way ANOVA indicated in the legends was employed to confirm that the depicted differences were significant in multi-comparison testing. The errors in The authors regret that in the original version of