Euglena gracilis is one of the few permitted edible microalgae. Considering consumer acceptance, E. gracilis grown heterotrophically with yellow appearances have wider food industrial applications such as producing meat analogs than green cells. However, there is much room to improve the protein content of heterotrophic culture cells. In this study, the effects of nitrogen sources, temperature, initial pH, and C/N ratios on the protein production of E. gracilis were evaluated under heterotrophic cultivation. These results indicated that ammonium sulfate was the optimal nitrogen source for protein production. The protein content of E. gracilis cultured by ammonium sulfate increased by 113% and 44.7% compared with that cultured by yeast extract and monosodium glutamate, respectively. The manipulation of the low C/N ratio further improved E. gracilis protein content to 66.10% (w/w), which was 1.6-fold of that in the C/N = 25 group. Additionally, amino acid analysis revealed that the nitrogen-to-protein conversion factor (NTP) could be affected by nitrogen sources. A superior essential amino acid index (EAAI) of 1.62 and a balanced amino acid profile further confirmed the high nutritional value of E. gracilis protein fed by ammonium sulfate. This study highlighted the vast potency of heterotrophic cultured E. gracilis as an alternative dietary protein source. The global shortage of protein supply will be one of the biggest problems in the next 30 years, especially when the population is projected to exceed 9.1 billion, and the meat demand might rise to 455 million tons by 2050 . Th. Th22]. E. gracilis protein, the amino acid profiles under different nitrogen sources were determined. The samples fed with urea and sodium nitrate were not analyzed since these two nitrogen sources cannot be utilized by E. gracilis. As shown in E. gracilis protein had a balanced amino acids profile, including cysteine and methionine, which are typically insufficient in legumes, and lysine, which is the first limiting amino acid in cereals [E. gracilis protein. Glutamic acid served as a precursor of many amino acid anabolisms, such as transamination to form aspartic acid and alanine [Chlorella vulgaris, Dunaliella bardawil, S. obliquus, Arthrospira maxima, Spirulina platensis, and Aphanizomennon sp. [C. vulgaris protein [To evaluate the nutritional value and NTP of  cereals ,24. Furt alanine ,26. Thisnnon sp. . The MSG protein . HoweverAk values are similar among these three groups, ranging from 6.11 ± 0.01 to 6.17 ± 0. A previous study investigated the Pk value of 10 different microalgal species, which ranged from 3.60 ± 0.27 to 4.99 ± 0.64 [Pk and NTP value of E. gracilis sample fed with ammonium sulfate were 4.87 ± 0.13 and 5.52 ± 0.06, respectively. The Pk of E. gracilis protein was comparable with most of the reported species but were shown to be higher than Skeletonema costatum (3.82 ± 0.33), Prorocentrum minimum (3.88 ± 0.68), and Dunaliella tertiolecta (3.99 ± 0.48) [NTP value of four industrial-applied microalgae  were recalculated. The results showed that the NTP (ranging from 4.68 to 5.35) and Pk (ranging from 3.00 to 4.43) values of these four microalgae were all lower than those obtained from the present study [E. gracilis has a lower non-protein–nitrogen content than microalgae species [E. gracilis, when fed with ammonium sulfate, could be considered a reliable and promising protein source.Based on the amino acid profile, NTP was calculated since the conventional approach of using a versatile NTP value of 6.25 may lead to an overestimation of the protein content as the presence of non-protein nitrogen compounds, such as inorganic nitrogen, nucleic acids, and amino sugars . The kA 9 ± 0.64 . The kP  ± 0.48) . Recentlnt study . This in species . These fE. gracilis was found to grow well under an initial pH of 3.5–7.5  and 32 °C (53.05 ± 0.89%), which were higher than 25 °C (46.83 ± 0.70%) and 35 °C (44.29 ± 2.56%). In addition, the protein yield of E. gracilis cultured at 25 °C (2.32 ± 0.02 g/L), 28 °C (2.23 ± 0.04 g/L), and 32 °C (2.24 ± 0.10 g/L) was significantly higher than that cultured at 35 °C (1.61 ± 0.11 g/L)  D, implyiat 32 °C . ConsequE. gracilis and further excavate its protein accumulation potential, the influence of the C/N ratio on protein biosynthesis in E. gracilis was studied. E. gracilis showed a remarkably high protein content of 66.10 ± 1.12%, which is comparable to that of traditional microalgal protein sources such as C. vulgaris (51–58%) and A. maxima (60–71%) [E. gracilis was increased by 169% under C/N = 10. This emphasizes the enormous influence of the nitrogen source and C/N ratio for E. gracilis protein production. This result also confirmed the great potential of heterotrophic E. gracilis as a dietary protein source. However, the relationship between nitrogen availability and protein content was not always positively correlated. The C/N = 5 group displays lower protein content compared with the C/N = 10 group  [To find out the impact of the C/N ratio on (60–71%) . Compare10 group A, while 10 group B. A simient drop . Additiogracilis . The higecreased . The aci00 mg/L) .E. gracilis cells with an increase in the C/N ratio from 10 to 25  and C/N = 10 (4.83 ± 0.06 g/100 g protein) and in tyrosine between C/N = 10 (4.33 ± 0.87 g/100 g protein) and C/N = 25 (3.21 ± 0.06 g/100 g protein), which implied that the variation of C/N ratio did not significantly alter amino acid profile of E. gracilis  changed with C/N ratio , the amigracilis . The sta/N ratio .NTP. The Pk and NTP values were at a similar level among the five groups. The C/N = 5 group had a significantly higher Ak value of 6.26 ± 0.07 than the C/N = 25 group of 6.17 ± 0.01  and Spirulina sp. (EAAI = 1.63), which are the conventional microalgal protein source [D. salina protein (EAAI = 1.53), which is also a novel microalgal protein source [E. gracilis was a promising single-cell-protein resource with high protein content and nutritional value.The consistent amino acid pattern observed in different C/N ratio groups resulted in a stable 7 ± 0.01 . In addi quality . This wan source , while in source . These rEuglena gracilis Klebs (CCAP 1224/5Z) was purchased from the Culture Collection of Algae and Protozoa . Algal cells were maintained on an agar plate with modified Hutner’s medium at 16 °C [E. gracilis was inoculated into 20 mL modified Hutner’s medium in a 50 mL Erlenmeyer flask and cultivated in shakers  in darkness for 5 days. The algal liquid was subsequently inoculated (7.5% v/v) into a 250 mL Erlenmeyer flask with a total volume of 100 mL and shaken for 48 h in the same condition for further use as a seed.at 16 °C . For eacSequential optimization experiments of the nitrogen source , temperature , initial pH , and C/N ratio  were conducted in 250 mL Erlenmeyer flasks with a working volume of 100 mL. The initial biomass concentration was kept around 0.4 g/L for each inoculation. The initial glucose concentration was fixed at 10 g/L (except for the sodium glutamate group to maintain the consistent C/N).Five microliters of cell culture were sampled and washed twice before being filtered on weighted GF/C film  and dried in the 80 °C vacuum dryer overnight to measure the biomass concentration. The samples were collected for further analysis 12 h after they reached the stationary phase. All the sample was washed three times with deionized water and freezing dried.E. gracilis was calculated with a conversion factor of 6.25 [The crude protein content of the sample was measured using the Dumas method . Around  of 6.25 .662, Ab646, and Ab480 are the sample absorbances at 662, 646, and 480 nm, respectively.Chlorophyll a, chlorophyll b, and carotenoid content were determined using a spectrophotometer at the wavelength of 480, 646, and 662 nm . PigmentThe amino acid composition analysis was proceeded by acid hydrolysis except tryptophan. Around 50 mg of lyophilized sample was acid hydrolyzed by 6 M HCl at 110 °C for 24 h under vacuum conditions. The sample was cooled down and neutralized via saturated sodium carbonate until the pH reached 7. The hydrolyzed sample (10 μL) was mixed with 70 μL of AccQ-Tag buffer and 20 μL of derived AccQ-Tag reagent solution . The mixFor tryptophan analysis, 50 mg of the sample was hydrolyzed by 5 M LiOH at 110 °C for 24 h under vacuum conditions. After hydrolysis, the sample was cooled down and neutralized by 6 M HCl until the pH reached 7. The neutralized sample was filtered via filter paper and analyzed using a Waters ACQUITY ultra-performance liquid chromatography–mass spectrometry (UPLC-MS) equipped with a PDA detector and BEH C18 column  .Ei, ΣDi, and N% are the summation of anhydrous amino acid, the summation of the nitrogen content of each anhydrous amino acid, and the nitrogen content of E. gracilis sample determined by the Dumas method using the conversion factor of 6.25, respectively [Ak and Pk are the upper bound and lower bound of the conversion factor, respectively [NTP, the average value of Ak and Pk, is considered the best conversion factor for practical use [The NTP was calculated according to the equation below .kA = Eiectively ,45. kA aectively . NTP, thn was the essential amino acid (EAA) content over total protein in the sample (mg/g protein) and AAn was adult EAA content (mg/g protein) requirements issued by WHO [The EAAI was calculated according to the equation  as follod by WHO . The quad by WHO .v/v) sulfuric acid in methanol and methylbenzene with heptadecanoic acid  as the internal standard. Samples were incubated in a 50 °C water bath overnight and analyzed using a gas chromatography–mass spectrometry (GC-MS) equipped with a DB-WAX column (30 m × 0.25 mm × 0.25 μm)  [Twenty micrograms of lyophilized samples were methyl esterized with 1%  .2SO4 for 1 h at 35 °C. Samples were then added to 10 mL of deionized water for further hydrolyzation at 98 °C for 1 h. The hydrolyzed samples were further determined using the phenol–-sulfuric acid method [Ten micrograms of lyophilized samples were hydrolyzed by 2 mL of 12 M Hd method .The freshly prepared samples were washed with PBS buffer twice and fixed overnight with 2.5% glutaraldehyde buffer at 4 °C. The samples were washed with 0.1 M PBS buffer (pH 7.0) followed by 1–2 h fixing with 1% osmic acid. The osmic acid was then replaced by 0.1 M PBS buffer (pH 7.0) and gradient dehydrated by a graded series of alcohol. After being replaced with acetone, the samples were embedded with epoxy resin and ultrathin sectioned to 70–90 nm thickness using an ultramicrotome EM UC7 . The ultrathin-sectioned samples were stained with acetic acid glaze and lead citrate and observed using TEM JEM1200EX  operated at 120 kV.p < 0.05.All experiments were conducted in triplicates. The results were expressed as mean ± standard deviation (SD). The statistical significance of the results was validated using a one-way analysis of variance (ANOVA) in the SPSS 26.0 software ; the significance level was set at E. gracilis growing heterotrophically. The low cultural C/N ratio condition was favorable for protein production, and the protein content could reach up to 66.10% (w/w). In addition, the protein quality evaluation of the heterotrophic E. gracilis protein showed a high NTP factor (5.63) and high EAAI (1.62), further indicating its high nutritional value. These findings suggested that heterotrophic cultured E. gracilis has great economic potential as an alternative protein source due to its high protein content and nutritional value, acceptable color, cell wall-free nature, and dietary-approved status.In the present study, we demonstrated that ammonium sulfate could significantly enhance the protein biosynthesis ability of


Various phenylisothiocyanates were added to solution of l-cysteine in methanol: water (1 : 1 v/v) at room temperature and stirred for 24 h. The precipitated solid was recrystallized from n-butanol. Pure compounds were characterized by NMR (1H and 13C), FTIR, and CHNS. Tertiary amine containing N-(4-(diethylamino)phenyl)-N′-(2-mercapto-carboxyethanyl)thiourea 17, N-(4-(dimethylamino)phenyl)-N′-(2-mercapto-carboxyethanyl)thiourea 16 and trimethoxy containing N--N′-(2-mercapto-carboxyethanyl)thiourea 14 were more active than galantamine against AChE and BChE enzymes. In tyrosinase enzyme inhibition activity, compound 14, 10, 12, 6, 13, and 11 exhibited higher tyrosinase inhibitory activity showing IC50 values of 1.1 ± 0.1, 1.5 ± 0.3, 1.6 ± 0.6, 1.9 ± 0.5, 2.2 ± 0.9 and 2.9 ± 0.2 mM, respectively. In urease enzyme inhibition activity assay, 17 showed higher activity. This work demonstrates the pharmacological significance of chiral thiourea derivatives synthesized from l-cysteine and shows their potential. There is a need to perform more in vitro and in vivo biological activities followed by clinical trials to bring such thiourea to the market.A series of chiral thioureas ( Expect few ribozymes, enzymes are protein-based biological catalysts. They catalyze chemical reactions in living things under moderate conditions with 100 % efficiency and no by-products. While biochemical transformations are easy and fast in the presence of enzymes, they do not take place, or it takes too long for them to occur in the absence of enzymes. Therefore, enzymes are essential biomolecules for the continuity of life ,,(1 – 17)1 [3 [16  from the(1 – 17) (compound 11 except), a weak band was observed in compound 11 at 3082–3357 cm−1 for NH bands attached to the aromatic ring and at 3003–3227 cm−1 for NH bands attached to aliphatic - CH - as expected. Additionally, the -OH band of the carboxylic acid group at 3659 – 3676 cm−1, aromatic C - H bands at 2961 – 3148 cm−1, aliphatic C - H bands at 2900 – 2969 cm−1, C = O band of the carbonyl group at 1568 – 1670 cm−1, NH band at 1477 – 1537 cm−1, C = S band of thiocarbonyl group at 1319 – 1420 cm−1, C - N band at 1228 – 1297 cm−1 and SH band of thiol group at 2509–2601 cm−1 were observed. The characteristic N - H, C = S and C - N tensile vibrations has been reported at 3190–3384, 1302–1393 and 1201–1282 cm−1, respectively [4, 5 and 9 containing fluorine were observed at 1208–1223 cm−1, C - Cl band of compound 5, 8 and 9 containing chlorine at 1081–1215 cm−1 while C - Br band of compound 2 containing bromine were observed at 1066 cm−1.In the IR spectra of chiral thioureas 1H NMR spectra of the synthesized compounds (1 – 17), -NH peaks linked to the aromatic ring of the thiourea ( - NH - CS - NH - ) group were observed at 7.13–9.61 ppm, while the - NH peaks linked to the aliphatic structure were observed at 7.71–10.55 ppm. Proton of carboxylic acid ( - COOH) was observed at 9.70–11.72 ppm; - CH - proton as multiple at 4.78–5.38 ppm; and the thiol ( - SH) proton were found at 1.08–3.17 ppm. - CH2 - protons resonated as Ha and Hb at 2.91–3.85 and 2.81–3.64 ppm, respectively. Protons in the - CH2 - SH are neighboring the chiral carbon, which are heterotopic as reported in our previously study [1HNMR peaks of all the synthesized compounds are given in In the ly study . Detaile17, 16 and 14 exhibited excellent activities than galantamine in both assays. Moreover, against AChE, 17 (IC50: 5.7 ± 1.0 μM), 10 (IC50: 6.8 ± 1.1 μM), 9 (IC50: 7.2 ± 0.5 μM), 12 (IC50: 7.8 ± 0.6 μM), 6 (IC50: 8.1 ± 0.9 μM), and 8 (IC50: 9.5 ± 1.2 μM) compounds exhibited higher activities. In the BChE inhibitory assay, 17 (IC50: 18.1 ± 0.5 μM), 16 (IC50: 24.6 ± 0.7 μM), 14 (IC50: 29.5 ± 1.1 μM), 13 (IC50: 37.2 ± 0.1 μM) and 10 (IC50: 45.8 ± 0.4 μM) displayed higher activities than galantamine (IC50: 46.4 ± 0.8 μM).All the synthesized compounds were tested for their in vitro anticholinesterase inhibitory activity against AChE and BChE enzymes. The results are compared with galantamine as given in l-mimosine as given in 14, 10, 12, 6, 13, and 11 showed the best tyrosinase inhibitory activity with IC50 values of 1.1 ± 0.1, 1.5 ± 0.3, 1.6 ± 0.6, 1.9 ± 0.5, 2.2 ± 0.9 and 2.9 ± 0.2 mM, respectively.Synthesized chiral thiourea derivatives were evaluated for their inhibitory effects on tyrosine enzyme at different concentrations. The results were compared with kojic acid and 14 (IC50: 13.4 ± 0.8 μM), 17 (IC50: 16.5 ± 0.6 μM), 10 (IC50: 20.9 ± 1.0 μM), and 11 (IC50: 22.1 ± 0.1 μM) exhibited excellent activities in the urease inhibitory activity than thiourea with IC50 of 24.20 ± 0.3 μM.The chiral thiourea derivatives were evaluated for their inhibitory effects on urease enzyme at different concentrations. The results were compared with thiourea . Compoun(1 – 17) were computed using SwissADME online toolkit. Evaluated parameters are presented in in silico study determines the drug-likeness of synthesized compounds compared to known drug. In 14. A good oral bioavailability is evaluated as the main of Log P ( < 5), MW ( < 500), HBA ( ≤ 10) and HBD ( < 5) values. The molecular flexibility is identified by the number of rotatable bonds (nROTB) which must be <10. nROTB values for the synthesized chiral derivatives are in the range of 6–9. Solubility parameter is whether the drug is soluble or moderately soluble, an important factor for the absorption of the drug. The insolubility is calculated using log S (ESOL) ranges 0–6 [−130 of molar refractivity, 180–480 of molecular weight and 20–70 of number of atoms. According to the Egan rule, which put forward by Pharmacia Company for the prediction of human intestinal absorption stating, the log P value should not be more than 5.88 while TPSA should not be more than 131.6. The orally active stated by Verber for GSK pharmaceuticals, particularly, in some drugs like steroids, the molecular weight should be more than 500 D and nROTB number 10 or fewer, while PSA should not be more than 140 A0. Parameters of the Muegge rule proposed by Bayer Pharmaceuticals should be Mw (200–600 D), log P (−2–+5), TPSA ( < 150), nROTB ( < 15), HBD ( < 5), and HBA ( > 10) and, < 7 number of rings, > 4 number of carbon atoms, and number of heteroatoms more than 1. PAINS in In the development the oral bioavailability of new drug candidates, obtaining the maximum level of bioavailability is thought to play a significant role , 41. Mannges 0–6 . Pharmacin silico prediction to use ADME  prediction that brings us advantages over experimental predictions. For this purpose, the ADME studies of synthesized chiral thiourea compounds were carried out and are shown in −130A), saturation (0.25 < 1), size (150 g/mol > 500g), and solubility ( < 6).These studies give us important information without conducting experimental studies on the possible effects of chemical compounds on metabolism from their molecular structure and whether they can be used as drugs. Thousands of molecules are synthesized in the world every year. If will be very costly, is we do their bioactivity tests in the laboratory environment. Therefore, ADME studies are very important in understanding the potential of compounds as drugs . It playBrain Or IntestinaL EstimateD permeation (BOILED - Egg) method is handled by calculating the polarity and lipophilicity of small molecules. This approach is widely used to provide a visual cue in profiling synthesis of novel compounds in terms of their potential to be orally absorbed [via passive diffusion to finally place the BOILED - Egg (Brain or Gut Prediction model of Change). The graphical prediction of GI and BBB permeation of the synthesized thiourea compounds is shown in 7, 10, 11, 12, 13, and 14 except) lie in this white ellipse. Compounds 7, 10, 11, 12, 13, and 14 were in the gray area showing poor intestinal absorption. The blue dots, evidence of exhibiting good bioavailability, indicated that compounds could be a substrate for P - glycoprotein, reducing its absorption and penetration within brain. Particularly, compounds 1, 2, 3, 4, 5, 15, 16 and 17 may be promising agents that can be absorbed very easily by the gastrointestinal tract without a potential BBB permeability.Gastrointestinal absorption (GI) and blood-brain barrier (BBB) are two pharmacokinetic behaviors critical to predict at different stages of the processes of drug discovery. As an accurate predictive model, the absorbed , 45. For(1 – 17) is being reported in this study for the first time. Generally, these compounds exhibited excellent anticholinesterase, tyrosinase and urease inhibition activities. Tertiary amine  and trimethoxy (14) based compounds showed more activity than galantamine against both AChE and BChE enzymes. According to the obtained data, the chiral thiourea derivatives containing a tertiary amine and trimethoxy groups utilized for their AChE and BChE inhibitory activity may be promising for further studies to treat Alzheimer’s disease.Alzheimer’s is an irreversible brain disorder defined by the loss of memory and learning ability in older patients. This disease is influencing large population around the world. Most of the clinically used drugs used to treat Alzheimer’s disease are acetylcholinesterase inhibitors (AChEIs). The enzyme Acetylcholinesterase (AChE), (E.C.3.1.1.7) have a key role in the hydrolysis of the released acetylcholine neurotransmitter . If acetMelanin is a group of natural pigments that plays primary role in determining the color of eye, hair, and skin. Congenital tyrosinase deficiency causes melanin production disorder in the body while skin defects happen due to excess melanin synthesis. Cancer and Parkinson’s disease depends on abnormalities in activity of tyrosinase –51. In tHelicobacter pylori (HP), which permits bacteria to survive at acidic pH of the stomach during colonization and consequently plays a significant role in the gastric and peptic ulcer pathogenesis [Urease  is a nickel-containing enzyme that catalyzes the urea hydrolysis into ammonia and carbamate, which is the last step of nitrogen metabolism in living organisms , 58. Theogenesis .N-(substitutedphenyl)-N′-(2-mercapto-carboxyethanyl)thioureas derivatives (1–17) were synthesized in this study. Structures of synthesized compounds were confirmed by spectroscopic methods using IR, 1H NMR, and elemental analysis . Compounds containing tertiary amine in the synthesized thiourea derivatives, especially those containing 2,4-diomethoxy, 2,5-dimethoxy, and 3,4,5-trimethoxy, showed notable urease activities. In conclusion, novel thiourea-derived pharmaceuticals can be synthesized for urease inhibition based on tertiary amine and methoxy groups.A series of chiral Figure S1The bioavailability radar of synthesized chiral thioureas and enzyme inhibitor standards using Swiss ADME predictor.


We have investigated the mechanical properties focusing on Debye temperature (ΘD), thermal conductivity (κ), Grüneisen parameter (γα), dominant phonon wavelength (λdom), and melting temperature (Tm). We find that the lattice thermal conductivity is significantly reduced when GeTe is alloyed into the following compositions: Ge0.75Sb0.13Pb0.12Te, Ge0.61Ag0.11Sb0.13Pb0.12Bi0.01Te, and Ge0.61Ag0.11Sb0.13Pb0.12Mn0.05Bi0.01Te. This reduction is due to the mass increase and strain fluctuations. The results also show that Ge0.61Ag0.11Sb0.13Pb0.12Bi0.01Te solid solution has the lowest Young’s modulus (30.362 GPa), bulk and shear moduli (18.626 and 12.359 GPa), average sound velocity (1653.128 m/sec), Debye temperature (151.689 K), lattice thermal conductivity (0.574 W.m–1.K–1), dominant phonon wavelength (0.692 Å), and melting temperature (535.91 K). Moreover, Ge0.61Ag0.11Sb0.13Pb0.12Bi0.01Te has the highest Grüneisen parameter with a reduced and temperature-independent lattice thermal conductivity. The positive correlation between ΘD and κ is revealed. Alloying of PbSe-based high-entropy by Sb, Sn, Te, and S atoms at the Se and Pb sites resulted in much higher shear strains resulted in the reduction of phonon velocity, a reduced ΘD, and a lower lattice thermal conductivity.GeTe-based and PbSe-based high-entropy compounds have outstanding thermoelectric (TE) performance and crucial applications in mid and high temperatures. Recently, the optimization of TE performance of high-entropy compounds has been focused on reducing thermal conductivity by strengthening the phonon scattering process to improve TE performance. We report a first-principles investigation on nine GeTe-based high-entropy chalcogenide solid solutions constituted of eight metallic elements  and 13 PbSe-based high-entropy chalcogenide solid solutions: Pb The atomic concentration of elements in high-entropy (HE) materials can be between 5 and 35% of the samples3. The concept of HE originated from the hypothesis that the solid solution is stabilized by a high configurational entropy of mixing4. Alternative criterion to define a HE material is the value of entropy of mixing ΔSmix expressed as R, N, and xi are the gas constant, number of components, and atomic fraction of the components, respectively5. Materials can be classified as low-entropy (ΔSmix less than 0.69R), medium-entropy (ΔSmix between 0.69R and 1.59R) and high-entropy (ΔSmix larger than 1.60R)6. The synthesis of HE materials is an emerging field of research which aims to design multicomponent single-phase materials. The multicomponent materials contain a minimum of 5 elements in nearly equal atomic ratios or not so equal in high-entropy metal alloys (HEMAs). HEMAs have a wide range of remarkable mechanical8, dielectric9, and superconducting10 properties. Recently, researchers found that HE materials can be disordered with severe lattice distortions and non-diffusion characteristic of the atoms contained therein exhibiting low thermal stability11. The scattering of phonons increases due to the severe lattice distortion, thus reducing lattice thermal conductivity of HE materials12. Single phase HEAs possess high hardness13, high strength14, good wear15, and erosion resistance16. Other multicomponent compounds, such as high-entropy oxides (HEOs)18, borides19, and carbides21 have also been synthesized recently.High-entropy alloys (HEAs) are alloys with high configuration entropy obtained by increasing the number of constituting elements , selenium (Se), and sulfur (S). In particular, the family of IV − VI compounds have attracted a great attention due to their optoelectronic, microelectronic, and high thermoelectric (TE) performance30. Recently, the most investigated category of IV − VI semiconductor chalcogenides are: SnS31, SnSe32, SnTe33, GeSe34, GeTe35, PbS36, PbSe37 and PbTe38.Despite the intense research on HE materials, HE materials with semiconducting characteristics have not yet been explored. HE materials having a band gap have a wide range of electronic, thermoelectric, and optical applications39. The performance of TE devices is described by the parameter ZT called figure of merit defined as ZT = S2σT/κ, where σ is the electrical conductivity, S is the Seebeck coefficient, T is the absolute temperature, and κ is the thermal conductivity. To improve the TE performance of chalcogenides, doping and alloying have been applied recently. For example, TE performance of Bi23, Sn, and GeTe alloys has shown to be enhanced44. The attempts to improve TE performance, by increasing TE power factor (PF) and lowering the lattice thermal conductivity (κL) have skyrocketed. Other attempts include introducing the configurational entropy via doping or alloying techniques45. For examples, chalcogenide compounds Ge0.84In0.01Pb0.1Sb0.05Te0.997I0.00329, PbSnTeSe46, and (Sn0.7Ge0.2Pb0.1)0.75Mn0.275Te47 achieved the enhancement of TE performance. Apparently, this strategy has been successful48. The sluggish diffusion and sever lattice distortion49 in HE materials have induced many fascinating TE properties such as low κL and very high ZT51. HEA are widely used to enhance TE performance in many chalcogenide systems such as (SnGePbMn)Te47, BiSbTe1.5Se1.552, and (PbTe)1−2x(PbSe)x(PbS)x53. A high figure of merit (ZT = 1.42 at 900 K) was achieved47 in (Sn0.74Ge0.2Pb0.1)0.75Mn0.275Te.The direct conversion of heat into electricity under a temperature gradient is carried out by thermoelectric (TE) devices. TE technology is one of the promising green solutions to mitigate the energy and environment crisis54 is a promising semiconductor with a narrow band gap and good TE performance. GeTe belongs to the IV-VI group and has rhombohedral crystal structure (R3m) at room temperature56. It exhibits a sudden phase transition to the cubic rock-salt structure (Fm-3 m) at around 700 K57. Compared to PbTe and SnTe, GeTe is much less explored. Pure GeTe has average TE properties with maximum ZT of less than 0.8 at 720 K58. Electronic structure, dynamical, dielectric, and elastic properties of pure GeTe was investigated using density functional perturbation theory by R. Shaltaf et al.59. They used the Hartwigsen-Goedecker-Hutter pseudopotentials including spin–orbit coupling to achieve high accuracy of the calculations. They found that GeTe is a semiconductor with a direct energy band gap of 0.48 eV. However, alloyed and doped rhombohedral crystals of GeTe with high TE performance have been reported61 revealing its reduced lattice thermal conductivity and higher ZT (ZT ≈ 2.2). This was achieved in Ge1–x–ySbxInyTe62, Ge1–x–ySbx ZnyTe63, and Ge1–x–yBixCdyTe64. Other attempts were carried out to reduce the phase transition temperature of GeTe by dual-doping of Sb and Mn57, and Bi and Mn65. Thus, TE performance of alloyed and doped GeTe was enhanced in the medium temperature range of 700 K. By introducing Ag, Sb, Pb, and Bi to Ge sites to form a high-entropy GeTe-based material, higher electrical transport and lower thermal conductivity were achieved by Jiang et al.66.Among chalcogenide materials, germanium telluride GeTe70. They belong to the IV-VI group and have a simple NaCl-type (B1) structure at ambient conditions. They are narrow gap semiconductors having low thermal conductivities even at high temperatures (500–700 k)71. This makes them good TE materials. TE performance of PbSe was enhanced by doping with elements such as B, Ga, In, Tl, Pb73, Al74, Sb75, and Bi76. HE technique has also been used to achieve high TE performance of PbSe-based HE compounds. Jiang et al. studied TE performance of Pb0.89Sb0.012Sn0.1Se0.5Te0.25S0.2577, Pb0.975Na0.025Se0.5S0.25Te0.25, and Pb0.935Na0.025Cd0.04Se0.5S0.25Te0.25 solid solutions78. They exhibit high ZT and low thermal conductivity.Lead chalcogenides PbX  have been extensively studied in the last decades due to their applications as TE materials available at medium temperature (400–900 K)κL) has skyrocketed. Creating a strong phonon scattering via phonon engineering is one of the best ways to minimize κL. This can be achieved by replacing light atoms by heaver atoms80, creating point defects81, enhancing strong lattice anharmonicity82, and producing dislocations83. Thus, manipulation of the phonon dispersion is the key to reduce κL. The phonon dispersion in can be expressed as in Eq.  which results in a low κL. Anharmonic lattice vibrations (anharmonicity) is connected to F which can be manipulated by using the elastic lattice strains, or the change to non-equilibrium atomic positions from its equilibrium position. While M can be changed through substitution, removal, or insertion of atoms. Clearly, a significant reduction in κL can be carried out by creating a large change in M. In the current study, we focus on the large change in M through alloying to create a significant reduction in κL.Obviously, optimization of TE performance via the minimization in lattice thermal conductivity  calculations were carried out for small models with few atoms (16 atoms) by using special quasi-random structure (SQS)85. They calculated several mechanical parameters such as Young's modulus, shear modulus, Poisson’s ratio, and Grüneisen parameter. The purpose of using SQS approach is to simulate a random solid solution using a relatively very small supercell to reduce the computational cost. However, to efficiently mimic the chemical disordering of multi-principal element in a random solid solution, using a large cell is required. Jiang et al. missed analyzing the effect of Grüneisen parameter on lattice thermal conductivity and calculating several crucial parameters such as Kleinman parameter, machinability index, Debye temperature, minimum thermal conductivity, and melting temperature.There are recent studies of GeTe-based HE0.99-ySb0.012SnySe1-2xTexSx  and Pb0.99-ySb0.012SnySe1-2xTexSx  as listed in Fig. R3m) using the supercell (SC) method86. Elements Ag, Sb, Pb, Bi, Cd, Cu, Sn, and Mn are distributed randomly in these nine HE models (m1 to m9). For PbSe-based HE models, thirteen solid solutions are modeled from single-phase rock-salt structure (space group fcc) with supercells each consisting of 1000 atoms. Next, the elements Sb, Sn, Te, and S are distributed randomly in these thirteen HE models. Large supercell for such HE chalcogenides are constructed for the first time in this work. Our calculations are performed using DFT-based package, Vienna Ab initio Simulation Package (VASP). The current methods do not consider the effect of temperature. Nevertheless, the configurational entropy is captured using randomly generated supercells. More details of the computational methods used can be found in the supplementary information (SI). The results are presented in “0.61Ag0.11Sb0.13Pb0.12Bi0.01Te (m5) with their solid solution composition and model numbers in the box at right, and (b) shows Ball and stick structure of Pb0.99Sb0.012Se0.5Te0.25S0.25 with their solid solution composition in the box on the right.In this study, we expand Jiang et al.’s work to fill the gap by investigating the elastic and thermal properties of pure GeTe(m0) and nine randomly disordered solid solutions of GeTe-based HE chalcogenides. They are listed in Fig. ij) are listed in Table ij values, m0 and nine solid solutions fulfill the mechanical stability criterial for trigonal structure87  and (001) planes, respectively. Large values of C11, C22, and C33 indicate incompressibility under uniaxial stress along x, y or z axes, respectively.The elastic constants correlate stress to strain behavior. Elastic constants describe the response of materials under external forces. In this work, we calculated the mechanical properties of m0 and the nine GeTe-based high-entropy solid solutions models using VASP. Details of the method are described in the SI. The calculated elastic constants  plane is easier relative to the shear along the (001) plane. C44 is higher than C66 in m2 and m9 indicating that the shear along the (001) plane is easier relative to the shear along the (100) plane. C66 is higher than C44 in m4, m6, and m7. A low value for C44 indicates a high shearability. Due to the lowest C44 value, m5 has the highest shearability among all other solid solutions. The values of the elastic constants generally depend on the lattice parameters and the strength of the bonds between the elements. Comparing m0 (pure GeTe) with models m1 to m9, we see that introducing Ag, Sb, Pb, Bi, Cu, Mn, Cd, and Sn elements at the Ge sites led to reduction in C11 and C22. Introducing Sb and Pb in m2; Ag, Sb, and Pb in m4; Ag, Sb, Pb, and Bi in m6; Ag, Sb, Pb, Bi, and Cu in m7; Ag, Sb, Pb, Bi, and Sn in m9 all resulted in higher value of C33. Introducing Ag, Sb, Pb, and Bi in Ge sites of m5 results in much reduced elastic constants in comparison with m0, which implies that the elastic strains in m5 are much higher. Reduction of elastic strains influences their thermal conductivity.Our calculations show C89 can be used to determine the contribution of the bond bending or the bond stretching/contracting to resist the external stress. More information about the formula used to calculate ζ is shown in Eq. n in Eq. . Kleinmaij and the compliance tensor Sij (Sij = 1/Cij) using Voigt–Reuss–Hill (VRH) approximation91 for poly-crystals. They are listed in Table The other mechanical parameters such as bulk modulus (K), Young’s modulus (E), shear modulus (G), and Poisson’s ratio (η) are obtained from the elastic coefficient Cij Sij = /Cij usinC44 can control the machinability also known as machinability index (µM) shown in Eq.  is less than 0.26, the material tends to be brittle otherwise, it is ductile. Hence, Frantsevich’s rule and Pugh’s criterion are equivalent.Machinability is the ease with which the materials can be machined at a low cost. The bulk modulus K together with n in Eq. . CalculaHV) formula to calculate the hardness (HV) of m0 and other nine solid solutions models. Vicker’s hardness formula was formulated by Chen et al.96 and Tian et al.97. Vicker’s hardness formula was derived from the elastic constants100 using Eq.  measurements are more suitable103. A semi-empirical formula that can estimate the micro-hardness (H) is listed in Eq. (H are listed in Table HV) and micro hardness (H) have the same trend and both of them are crucial parameters to be calculated for the chalcogenide models. From the above results, introducing Ag, Sb, Pb, Bi, Cu, Mn, Cd, and Sn elements at the Ge sites results in reduced brittleness and hardness of the nine solid solution models.We also used Vicker’s hardness or macro hardness . Lame’s constants can be used to measure the compressibility and the shear stiffness of a material, respectively. In most cases, and in the context of elasticity, μ carries the same information as shear modulus. The formula used to calculate λ and μ can be found in Eqs.  in Eqs.  and (S15AU) calculated using formula  and shear (Ashear) listed in Table  Acomp and Ashear have a value of zero, it indicates isotropic elastic behavior whereas their value of 1 indicates largest possible anisotropy. The shear anisotropic factor for {100} shear planes between the [011] and [010] directions (A1), the shear anisotropic factor for {010} shear planes between the [101] and [001] directions (A2), and the shear anisotropic factor for {001} shear planes between the [110] and [010] directions (A3) are calculated by the formulae  a aAU) calformulae . The val108 in Figs. The directional dependent mechanical properties, E, K in terms of compressibility (1/K), G, and η, are presented in three-dimensional (3D) surface plots via ELATE programΘD) originates from the theory of thermal vibration of atoms. ΘD in condensed materials can reflect the strength of covalent bonding and other thermal characteristics such as specific heat, melting temperature, and thermal expansion. ΘD is positively correlated with thermal conductivity (κ) and is an important parameter in high temperature applications. Anderson’s method is one of the straightforward and accurate methods to calculate ΘD. The formula used to calculate ΘD is shown in Eq. , the transverse (shear) velocity (vs), and the longitudinal sound velocity (vl) are shown in Eqs.  in comparison to pure GeTe (m0). m2 and m5 have the smallest values of νs. This significantly dampened transverse phonon modes (νs) would strengthen the scattering of phonons111 which in return results in a reduced lattice thermal conductivity (κL). Impedance parameter (Z) is used in acoustic applications such as noise reduction and transducer design. In this work, Z is calculated using Eq.  a aΘD) ori in Eqs.  respectising Eq.  and listκ) measures the performance of heat transfer at high temperatures112. It is crucial to calculate the minimum thermal conductivity (κmin) and lattice thermal conductivity (κL) since they determine the TE performance. Clarke’s model113, Cahill’s model114, and Slack’s model115 were used to estimate κmin and κ of m0 and other nine HE chalcogenide solid solutions at 300 K. The formulae  respectively. They are listed in Table κmin according to Clarke’s model and Cahill’s model, while m2, m5, and m8 have the lowest and comparable values of κ according to Slack’s model. The Grüneisen constant 116. If γα is large, the anharmonic vibrations are strong, indicating a small influence of temperature increase on the lattice dynamics and thermal properties. The calculated γα for m0 and other nine HE chalcogenide solid solutions are summarized in Table γα, m0 and m5 are the highest influenced under high temperature  whereas m2 with highest γα is less influenced under high temperature . However, strong anharmonic vibrations (higher γα) also indicate higher phonon scattering and thus low κ. Hence, according to this criterion, m5 should have the highest value of γα among other models. Another formula that estimates Grüneisen parameter using the Poisson’s ratio is shown in Eq. (vs) and the longitudinal (vl) sound velocities as shown in Eq. , , κ) measformulae  and S31κ measuren in Eq. . Poissonn in Eq. . vs and vl and vs   vl and ving Eqs. ) are lissing Eq. ) are lisvl/vs versus Grüneisen parameter, showing that Gruneisen parameter is linearly proportional to vl/vs. The increasing vl/vs on x-axis implies a decrease in vs. The decrease in vs from the softened transverse phonons is the reason for the increase of γα , which describes the interactions among the different branches of phonons, is largely strengthened, resulting in a temperature-independent and significantly reduced κL. Thermal conductivity (κ) calculated by Slack’s formula  shown in the sixth column of Table In Fig. f γα Eq. . Thus, tmula Eq.  consistsn in Eq. ). The A κ is mainly contributed by lattice thermal conductivity κL. κL has a relatively small values at low temperatures. κL has contributions from acoustic phonons (κa) and optical phonons (κo). It is crucial to calculate κL to ascertain whether the HE chalcogenides considered are good TE materials or not. We have calculated κL, κa, and κo using the Eqs. exp from Jiang’s work66 is listed in Table Tmelt) is correlated to ΘD, κL, and thermal expansion. Materials with low Tmelt tend to have lower ΘD and higher thermal expansion. For high temperature applications such as thermoelectric applications, it is crucial to identify the thermal limits or melting temperature (Tmelt) of a material. More information about the formula used to calculate Tmelt can be found in . α for m0 and nine solid solutions can be estimated from the formula (λdom).At low temperatures, the electron–phonon scattering is small. Thus, thermal conductivity the Eqs. , S36) a aκ is mathe Eqs.  respectifound in . The cal formula . Table Sλdom to identify the total energy of phonons or the maximum energy of phonons at a certain temperature. λdom and the mean free path (MFP) (the average distance that a phonon travels between two successive inelastic collisions) are positively correlated. These two parameters play a significant role in controlling κL. Shifting the heat phonon spectra towards shorter wavelengths  and shorter mean free paths may increase the scattering of phonons and reduce κL119. λdom can be roughly estimated at 300 K by using the formula . The calculated elastic constants of pure PbSe and eight solid solutions are listed in Table 11, C22, and C33 of Pb0.99-ySb0.012SnySe1-2xTexSx (with y = 0 and x = 0.25) or Pb0.99Sb0.012Se0.5Te0.25S0.25. With increase of x content, a gradual reduction of C44, C55, and C66 is observed in Fig. Tmelt) of both Pb0.99-ySb0.012SnySe1-2xTexSx and Pb0.99-ySb0.012SnySe1-2xTexSx. The theoretical density (ρ), the calculated sound velocity , ΘD, Tmelt, α, and λdom for Pb0.99-ySb0.012SnySe1-2xTexSx  and y = 0) solid solutions are listed in Table λdom has been shortened for all models. λdom has been shortened from its highest value(0.890 Å) in pure PbSe to its lowest value(0.718 Å) for Pb0.99Sb0.012Se0.5Te0.25S0.25 model. Shortening λdom reduces the mean free path of phonons which increases scattering of phonons resulting in a reduced κL.In this section, we focus on results of solid solutions Pbνm, ΘD. Tmelt, and α versus x content of Pb0.99-ySb0.012SnySe1-2xTexSx. νm, ΘD, and Tmelt decreases with increase of x content. The same trend is noticed for elastic constants. While thermal expansion increases with increase of x content, a sharp reduction in νm, ΘD, and Tmelt is notable for Pb0.99-ySb0.012SnySe1-2xTexSx (with y = 0 and x = 0.25) or Pb0.99Sb0.012Se0.5Te0.25S0.25 . A sharp increase of α is noted for Pb0.99Sb0.012Se0.5Te0.25S0.25. Lower νm and ΘD indicates weaker chemical bonds and a lower κL. The reduction in νm with increase of x content is large in the current study  and transverse (νs)) decrease.Figure κL and consequently enhancing the TE performance. Phonon velocities and κL are correlated by the Eq. (120:This can be understood via the correlation between the phonon velocity and the elastic constants shown in Eqs.  and 3)..3). Redu the Eq. 120:4\docγα. Figure 0.99-ySb0.012SnySe1-2xTexSx, which results in a reduced κL , Kleinman parameter(ζ), machinability index(µM), Cauchy pressures (CP), and Lame’s constants  for Pb0.99-ySb0.012SnySe1-2xTexSx solid solutions  and y = 0 are listed in Table vl /vs plot is shown in Fig. 0.99-ySb0.012SnySe1-2xTexSx versus x content. As can be seen, there is a gradual reduction of E, K, and G for all solid solution models with increase of x content with a notable sharp reduction with y = 0 and x = 0.25. On the other hand, Poisson’s ration increases with increase of x content. A gradual increase of the machinability index for all solid solution models with increase of x content, with a notable dip at 0.25 × content in Fig. λ) is larger than the second Lame’s constant (μ) for all solid solutions models indicating a higher compressibility than shear.The calculated Young’s modulus (E), bulk modulus (K), shear modulus (G), Poisson’s ratio (η), Pugh’s ratio (G/K), Vicker’s hardness (12–C44)121. The calculated CP of Pb0.99-ySb0.012SnySe1-2xTexSx versus x  and y = 0 are listed in Table 0.99-ySb0.012SnySe1-2xTexSx  or Pb0.99Sb0.012Se0.5Te0.25S0.25 which indicates dominant ionic bonds. The calculated AU, GV, GR, KV, and VR are given in Table AU of Pb0.99-ySb0.012SnySe1-2xTexSx varies from unity in different degrees. However, Pb0.99-ySb0.012SnySe1-2xTexSx  or Pb0.99Sb0.012Se0.5Te0.25S0.25 shows isotropic nature compared to other solid solutions.Pugh’s ratio (G/K) is also listed in Table 0.99-ySb0.012SnySe1-2xTexSx . The calculated elastic constants, Young’s modulus, bulk and shear moduli, Poisson’s ration, Vicker’s hardness, Kleinman parameter, machinability index, Cauchy pressure, Lame’s constants, and the universal anisotropic index of Pb0.99-ySb0.012SnySe1-2xTexSx  solid solutions are listed in Tables νm, ΘD, Tmelt, and α versus y content of Pb0.99-ySb0.012SnySe1-2xTexSx and x = 0.25. The first model: Pb0.99-ySb0.012SnySe1-2xTexSx  is discussed in the previous section. It is noticed that νm and ΘD have a sharp peak at y = 0.05 and then a gradual decrease with the increase in y content. A sharp reduction in Tmelt is notable for Pb0.99-ySb0.012SnySe1-2xTexSx (with y = 0.1 and x = 0.25). Thermal expansion coefficient has a sharp decrease at y = 0.05 and y = 0.1 and then gradually increases. Clearly, increasing y content while keeping x = 0.25 results in lower νm, ΘD, and Tmelt. The calculated thermal conductivities and Grüneisen parameter of Pb0.99-ySb0.012SnySe1-2xTexSx  with x = 0.25) are listed in Table ρ), νl, νs, νm, ΘD, Tmelt, α, and λdom(Å) for Pb0.99-ySb0.012SnySe1-2xTexSx  and x = 0.25) are listed in Table 0.99-ySb0.012SnySe1-2xTexSx has a notable dip at 0.1 y-content and then increasing rapidly with increase of y content. In Fig. κmin starts to increase till 0.05 of y-content and then gradually decreases thereafter. In Fig. κL starts to increase till 0.1 y-content and then decreases thereafter. y-content above 0.1 in Pb0.99-ySb0.012SnySe1-2xTexSx (x = 0.25) models results in much higher Grüneisen parameter and consequently, a reduced κL.In this section, we focus on results of solid solutions PbκL in most nine HE models under study. Furthermore, the alloy defects may have strengthened the phonon scattering, resulting in much depressed κL. Our results for κL show the same trend as the experimental data of Jiang et al.66. Materials with low ΘD and low thermal conductivity have remarkable applications as TE and thermal barrier coatings (TBC) materials. We revealed that there is a positive correlation between Debye temperature and thermal conductivity for the investigated nine HE models. A strong anharmonicity (high Grüneisen parameter) indicates weak atomic bonds, and thus low κL123. There is a positive correlation between sound velocity and the strength of interatomic interactions. Lower sound velocity indicates weaker interatomic interactions between atoms as indicated by large Grüneisen parameter124. We know from Eq. (κL can be reduced by minimizing three parameters: heat capacity (Cα), phonon relaxation time (τα), and phonon velocity (υ). If the speed of sound (i.e. lattice stiffness) can be manipulated via some methods, κL can be reduced significantly. One of the techniques for reducing speed of sound is inducing internal-strain fields, which are induced by lattice defects such as dislocations, alloying, and doping125. Internal- strains change the speed of sound which results in higher phonon scattering and lower κL. At small strains, the phonon frequency (ω) and the speed of sound, the Grüneisen tensor (γij), and the strain tensor (εij) are correlated by the following formula126:ε will reduce the phonon frequency and thus reduce κL. Compared to m0 (pure GeTe), the lattice parameters have been increased in m5 (Ge0.61Ag0.11Sb0.13Pb0.12Bi0.01Te) due to the alloying (see Table 0.61Ag0.11Sb0.13Pb0.12Bi0.01Cu0.003Te), subsequent to their suppression in m5. Multiple elements alloying introduces atomic disorder  in the HE model lattice, which in general is considered as point defects. Alloying also causes severe lattice distortion which together with the point defects are favorable for dislocation formation127. This results in a strong strain field to reduce lattice thermal conductivity. Dislocations can significantly change the mechanical properties. More dislocations in the lattice results in much lower Young’s modulus, bulk and shear moduli. However, Cu substitutional alloying in m6 may suppress the dislocation formation, resulting in higher elastic properties than m5.In GeTe-based HE models, Alloying by Pb and Bi atoms at the Ge atom sites produced higher mass and strain field fluctuations to scatter phonons due to the larger atomic size and heavier mass of Bi and Pb than Ge. This may be the main reason for the lower 0.99-ySb0.012SnySe1-2xTexSx. Rafal et al.116 has shown that enhancing bond anharmonicity by alloying of Pb1-xSnxTe results in larger γα which in return results in a reduced κL. We conclude that alloying with Sb, Sn, Te, and S atoms caused an increase of elastic strains and γα, a reduction of phonon velocity, Debye temperature, and consequently a significant reduction in κL. There is a dramatic decrease in the values of elastic constants  as x content increases to 0.25  in the values of elastic constants causes a dramatic decline of νs, νm, and θD of Pb0.99Sb0.012Se0.5Te0.25S0.25 . At x = 0.25, κ and κL have also the smallest values . After x = 0.25, the dislocation density starts decreasing and causing higher κL and elastic properties.In PbSe-based HE models, both strains and Grüneisen parameter have been increased through alloying by increasing x content of Pbsee Fig. a–c. This0.77Ag0.11Pb0.12Te(m1),Ge0.75Sb0.13Pb0.12Te(m2),Ge0.74Ag0.11Sb0.13Te(m3), Ge0.62Ag0.11Sb0.13Pb0.12Te(m4),Ge0.61Ag0.11Sb0.13Pb0.12Bi0.01Te(m5), Ge0.61Ag0.11Sb0.13Pb0.12Bi0.01Cu0.003Te(m6),Ge0.61Ag0.11Sb0.13Pb0.12Cd0.05Bi0.01Te(m7), Ge0.61Ag0.11Sb0.13Pb0.12Mn0.05Bi0.01 T(m8), and Ge0.61Ag0.11Sb0.13Pb0.12Sn0.05Bi0.01Te(m9) were investigated using first-principles calculations. The alloying of these solid solutions was carried out using the random solid solution model (RSSM) which is efficient for large supercells of 1080 atoms. Based on our calculations, m0 and all nine solid solution models are mechanically stable. m5 has the lowest Young’s, bulk and shear moduli. Generally speaking, random alloying of GeTe by Ag, Sb, Pb, Bi, Cu, Mn, Cd, and Sn elements at the Ge sites results in a reduced hardness, sound velocity, Debye temperature, and thermal conductivity. Among the nine solid solution models, m2, m5, m8 have the lowest Debye temperature and thermal conductivity. m1, m2, m5, and m8 have much lower lattice thermal conductivity compared to pure GeTe (m0). The models m2, m5, and m8 have the lowest and comparable values of κL  and Pb0.99-ySb0.012SnySe1-2xTexSx  are investigated in detail. The reduction in speed of sound and lattice thermal conductivity correspond to an increase in internal elastic strains. We conclude that alloying with Sb, Sn, Te, and S atoms caused an increase of elastic strains and γα, a reduction of phonon velocity, Debye temperature, and consequently a significant reduction in κL. Despite a large number of complex calculations on mechanical and thermal properties of very large supercells of high-entropy chalcogenides were done for the first time, there are obviously some drawbacks such as ignoring the lattice dynamics, However, phonon calculations is almost impossible to be carried out for such large supercells. We are encouraged by the current results and aspire to continue research in this direction for more complex and interesting high-entropy chalcogenides. It is desirable to improve the DFT calculations with better options, such as using either hybrid potential or Becke–Johnson potential. Finally, we believe our results can facilitate the design of new high-entropy chalcogenides with better thermoelectric performance.The elastic and thermal properties of pure GeTe(m0) and nine high-entropy models: GeVienna Ab initio Simulation Package (VASP)128. VASP is used to optimize the structures of solid solution models and calculate the mechanical properties. The one-electron orbitals are expanded in the plane wave basis set with an energy cut-off of 600 eV. The generalized gradient approximation (GGA) of Perdew, Burke, and Ernzerhof (PBE)129 is used as the exchange and correlation potential for solving the Kohn–Sham equation. The electronic and ionic force convergence criteria are set at 10−6 eV and 10−5 eV/Å respectively. The Monkhorst scheme130 is used with k-point meshes of 2 × 2 × 1 for GeTe-based HE solid solutions, while k-point meshes of 1 × 1 × 1 were used for all PbSe-based HE solid solutions. The stress vs strain scheme is used to calculate the elastic tensor. The following linear equation, also called the Hooke’s law, can be solved to obtain the elastic coefficient, Cij:σi is obtained by applying a strain єj of + 0.50% and − 0.50% to the equilibrium structure. More details on mechanical properties calculation are described in SI.Density functional theory (DFT) based method is utilized to perform the calculations implemented in the N in all ten models is calculated by the simple formula: N = 6 × (n2 × m), where the number 6 is the number of atoms in the GeTe simple cell. n and m are set to be 6 and 5, respectively, so N is 1080 atoms in the trigonal supercell. The lengths of the supercells equal to n × a and m × c, where a and c are the lattice constants of the simple trigonal GeTe crystal. RSSM method requires a large supercell with large number of atoms. A 1080-atom supercell can be considered to be the minimal size to justify the use of RSSM with high confidence. It is necessary to use “supercell” to capture different possible structural configurations of the HE models. In the present study, we assure that the statistical distribution of random distribution of alloying elements  is sufficient due to sufficiently large supercell with periodical boundary conditions that can account for the random distribution of the NN, second NN, and the third NN for each atom in the model. In compliance with the spirit of RSSM, it is carried out by writing a small script such that the atomic occupation of each site is completely random with no restriction to their NN atoms and beyond131. For PbSe-based HE models, the same procedure was followed, except that the formula of N was set to be: N = 8 × (n3), where the number 8 is the number of atoms in the PbSe simple cell and n is set to be 5. The lengths of the supercells equal to n × a where a is the lattice constant of the simple cubic PbSe crystal.GeTe-based supercells for high-entropy chalcogenide models in trigonal lattice are constructed based on the random solid solution model (RSSM). In RSSM method, the determination of the number of atoms, N, in the supercell is crucial. Supplementary Information.


Magnaporthe oryzae threatens rice production worldwide, and chemical control is one of the main methods of its management. The high mutation rate of the M. oryzae genome results in drug resistance, which calls for novel fungicide targets. Fungal proteins that function during the infection process might be potential candidates, and Mps1 (M. oryzae mitogen-activated protein kinase 1) is such a protein that plays a critical role in appressorium penetration of the plant cell wall. Here, we report the structure-aided identification of a small-molecule inhibitor of Mps1. High-throughput screening was performed with Mps1 against a DNA-encoded compound library, and one compound, named A378-0, with the best performance was selected for further verification. A378-0 exhibits a higher binding affinity than the kinase cosubstrate ATP and can inhibit the enzyme activity of Mps1. Cocrystallization of A378-0 with Mps1 revealed that A378-0 binds to the catalytic pocket of Mps1, while the three ring-type substructures of A378-0 constitute a triangle that squeezes into the pocket. In planta assays showed that A378-0 could inhibit both the appressorium penetration and invasive growth but not the appressorium development of M. oryzae, which is consistent with the biological function of Mps1. Furthermore, A378-0 exhibits binding and activity inhibition abilities against Mpk1, the Mps1 ortholog of the soilborne fungal pathogen Fusarium oxysporum. Collectively, these results show that Mps1 as well as its orthologs can be regarded as fungicide targets, and A378-0 might be used as a hit compound for the development of a broad-spectrum fungicide.Blast disease caused by  Magnaporthe oryzae  is the filamentous fungus that causes rice blast, one of the most devastating diseases of cultivated rice , foxtail millet , and grasses   , 4. It ie, etc. 57. To datM. oryzae is important for determining promising fungicide targets. M. oryzae disperses via conidia with wind and dewdrop splash  (www.frac.info/). Therefore, other kinases of fungal pathogens might be developed as novel fungicide targets.Kinases catalyze the transfer of the γ-phosphate of ATP to serine, threonine, or tyrosine residues of a substrate protein, resulting in signal transduction in eukaryotic living cells . In the .ac.uk/) . HoweverM. oryzae contains three mitogen-activated protein (MAP) kinases, Mps1, Pmk1, and Osm1  named DELopen (WuXi AppTec) see . Each co10.1128/mbio.02883-22.1FIG S1FIG S1, TIF file, 0.3 MB.Screening of small-molecule compound inhibitors with a DNA-encoded compound library. (A) Schematic diagram of DNA-labeled compounds of the DNA-encoded compound library. Each compound is linked to a DNA barcode, the sequence of which is used to identify the compound. (B) Inhibitor screening procedure with a DNA-encoded compound library. Download Copyright © 2023 Kong et al.2023Kong et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.02883-22.2FIG S2FIG S2, TIF file, 0.4 MB.SPR assay of the affinity between AMP-PNP and Mps1. (A) Comparison of the ATP and AMP-PNP chemical structures. The difference between them is highlighted in orange. (B) SPR sensograms. The results show that AMP-PNP at a concentration range between 15.625 μM and 500 μM does not interact with Mps1. Download Copyright © 2023 Kong et al.2023Kong et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vitro binding affinity (represented by the dissociation equilibrium constant [DK]) between A378-0 and Mps1 is 69.2 μM, confirming the interaction between them , with subtle modifications. The structure was determined at a 2.15-Å resolution. Each asymmetric unit contains one protein molecule. Residues Q5 to T360 and Q394 to D412 of Mps1 were built into the model, while others were not due to the invisibility of the corresponding electron density  and a larger C-terminal lobe , with a hinge region (residues E105 to E108) connecting them . The N-lExtra electron density was observed in the catalytic pocket, readily fitted with compound A378-0  and C. ABoth hydrogen-bonding interactions and hydrophobic interactions play roles in the interaction between A378-0 and Mps1. There are seven intermolecular hydrogen bonds between A378-0 and Mps1 . First, In addition to these directly formed hydrogen bonds between A378-0 and Mps1, there are water-mediated intermolecular hydrogen-bonding interactions that involve two water molecules. One water molecule mediates the interaction between the nitrogen atom of amide group II of A378-0 and three side chain oxygen or nitrogen atoms: oxygen atoms of residues D149 and D167 and the nitrogen atom of residue N154 . The othBesides hydrogen bonds, hydrophobic interactions also function in stabilizing A378-0 in the cleft between the N- and C-lobes of Mps1  and B. T10.1128/mbio.02883-22.3FIG S3FIG S3, TIF file, 1.4 MB.Hydrophobic interactions between A378-0 and Mps1. (A) Interactions calculated with LIGPLOT, with hydrogen bond information removed. Nonpolar or aromatic residues are shown in red and are regarded as residues for forming hydrophobic interactions with A378-0. (B) Three-dimensional view. A378-0 and Mps1 are shown and colored as described in the legend of Copyright © 2023 Kong et al.2023Kong et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the From the interaction analysis described above, we can see the two substructional groups of the phenyl ring of A378-0 participate in the formation of both hydrogen bonds and hydrophobic interactions with Mps1, which can explain why compound A581-0 exhibited a lower copy number and a lower enrichment value, i.e., a lower binding affinity, than A378-0 during library screening  to C.DK value of which exceeded the measurement limit of the Biacore 8k+ instrument. Therefore, it can be concluded that the four residues Q31, K52, E70, and D167 of Mps1 are important for its binding to A378-0, which also verified the accuracy of the Mps1/A378-0 structural model.We then carried out mutational studies of Mps1 to evaluate representative critical residues that are involved in interactions between A378-0 and Mps1 . Residue10.1128/mbio.02883-22.4FIG S4FIG S4, TIF file, 0.8 MB.SPR assay results for the interactions between A378-0 and Mps1 single mutants. The series of concentrations of A378-0 used are shown at the bottom of the charts. Download Copyright © 2023 Kong et al.2023Kong et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the M. oryzae virulence. Infection of plants by M. oryzae begins with appressorium development; i.e., M. oryzae conidia recognize and adhere to rice hydrophobic surfaces, germinate, and develop into dome-shaped infection cells, appressoria. Later, in the presence of gradually accumulating turgor pressure, a penetration peg is formed to penetrate the host cell wall, and the cells differentiate into primary hyphae and then invasive hyphae, which grow from cell to cell and result in visible lesions (M. oryzae (We then verified whether A378-0 possesses bioactivity in suppressing  lesions  9). Acc. AccM. o. oryzae .10.1128/mbio.02883-22.5FIG S5M. oryzae virulence and other processes. (A) A378-0 inhibits M. oryzae virulence on barley leaves. Isoprothiolane was used as a positive control. Bar = 1 cm. (B) Conidia per petri dish produced by M. oryzae on OTA plates in the presence or absence of A378-0. Error bars indicate standard deviations from three independent experiments. (C) Colonies formed by M. oryzae on OTA plates at 5 days postinoculation in the presence or absence of A378-0. (D) Sensitivity of M. oryzae to cell wall-digesting enzymes in the presence or absence of A378-0. Photos were taken after 1 h of treatment with 7 mg mL−1 lysing enzymes from Trichoderma harzianum. Bar = 20 μm. Download FIG S5, TIF file, 10.6 MB.A378-0 inhibits Copyright © 2023 Kong et al.2023Kong et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the M. oryzae conidia malonate] as a control. Isoprothiolane is a fungicide that suppresses phospholipid biosynthesis and can inhibit the appressorium penetration of M. oryzae at a concentration of 20 μg mL−1 , Slt2 from Colletotrichum gloeosporioides (accession number AFD31817.1), Mgv1 from Fusarium graminearum (accession number AAM13670.1), Mpk1 from F. oxysporum (accession number RKL49942.1), Smk3 from Sclerotinia sclerotiorum (accession number AOA52648.1), Pmk1 from M. oryzae (accession number XP_003712175.1), and Osm1 from M. oryzae (accession number XP_003714838.1). The sequences were aligned with Clustal Omega and viewed with MView. The red and cyan triangles indicate critical residues of Mps1 for forming hydrogen bonds and hydrophobic interactions with compound A378-0, respectively. The red rectangle indicates the hinge region, which is the boundary of the N-lobe and the C-lobe of these fungal MAP kinases. Download FIG S6, TIF file, 3.4 MB.Sequence alignment of Mps1 with its orthologs from fungal plant pathogens and the other MAP kinases of Copyright © 2023 Kong et al.2023Kong et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the os-2 gene), a MAP kinase that functions as a central signaling mediator during osmoregulation and responses to external stresses or stimuli of eukaryotic organisms . DRK also binds to the catalytic pocket of the kinase, similar to A378-0 (According to the Fungicide Resistance Action Committee (FRAC), there are seven fungicides that target kinases, which can be divided into two groups  Fungicides that target HOG1. (B) Fungicides that target Daf1. Download Copyright © 2023 Kong et al.2023Kong et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.02883-22.8FIG S8FIG S8, TIF file, 2.4 MB.Comparison of the CGD7/DRK, Mps1/A378-0, and ERK2/AMP-PNP complex structures. (A) Structural superimposition of these structures. The proteins and small-molecule compounds are shown as ribbons and sticks, respectively. (B) Close-up view of A378-0 and DRK from panel A. (C) Close-up view of AMP-PNP and DRK from panel A. In panels A and C, the magnesium ion in the ERK2/AMP-PNP complex that is essential for enzyme activity is shown as a lime sphere. Download Copyright © 2023 Kong et al.2023Kong et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The Mps1/A378-0 complex structure that we present in this study clearly elucidated the interactions between the two molecules, making the optimization of A378-0 more readily. In the structure, Mps1 adopts the αC-helix-in and DFG-aspartate-in conformations, both of which are necessary for a kinase to be in the active state  10). In. In10). 10.1128/mbio.02883-22.9FIG S9FIG S9, TIF file, 2.8 MB.Mps1 is in its active conformation in the Mps1/A378-0 complex structure. (A) Mps1 in the “αC-helix-in” and “DFG-aspartate-in” conformations. Both the side chains of residue E70 of the αC-helix and D167 of the DFG motif (green) point to the catalytic region. (B) The C-spine and R-spine of Mps1 have formed. Residues constituting the C-spine and R-spine are shown as spheres and are in green and gray, respectively. Download Copyright © 2023 Kong et al.2023Kong et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the M. oryzae. Pmk1 and Osm1 are the other MAP kinases of M. oryzae and share sequence identities of 51.4% and 39.8%, respectively, with Mps1 , we can see that the scaffold of A378-0 is absent from them, suggesting that the scaffold of A378-0 is novel and might serve as the basis for the development of new kinase inhibitors. A limitation of this research is that the chemical structure of A378-0 is more complex than those of the phenylpyrrole or dicarboximide fungicides that target two other MAP kinases, suggesting that substantial optimization is necessary to reduce the cost of the production of A378-0 derivatives  protein was expressed using Escherichia coli BL21(DE3). The E. coli strain was cultured in lysogeny broth (LB) medium supplemented with 100 μg mL−1 ampicillin. The coding sequence of Mps1 with an N-terminal 6×His tag was cloned into the pHAT2 vector  at an optical density at 600 nm (OD600) of 0.6, and the cells were cultured at 16°C for 20 h. Cells were harvested by centrifugation and resuspended in lysis buffer containing 20 mM HEPES (pH 7.3) and 400 mM NaCl. After sonication and centrifugation, recombinant protein in the supernatant was collected with nickel-charged resin and further purified with a Superdex 200 size exclusion chromatographic column (Cytiva) equilibrated with storage buffer containing 20 mM HEPES (pH 7.3), 150 mM NaCl, and 2 mM dithiothreitol (DTT). All of the purification procedures were performed at 4°C or on ice.The recombinant Mps1  three times and then heated at 95°C for 10 min to release compounds from Mps1 for the next round of screening. The above-described steps were repeated three times, and the compound solution obtained in the last round was used for PCR, sequencing, and analysis.Screening was performed according to the manufacturer’s suggestions, with subtle modifications. Briefly, nickel-charged resin was equilibrated with wash buffer . Twelve micrograms of Mps1 protein was captured with 40 μL of resin and divided in half: one half was used for the screening of the DNA-encoded compound library, and the other half was used as a negative control. The two samples were washed with selection buffer . The experiment was performed with the Biacore 8k+ instrument (Cytiva) at 25°C. Compound A378-0 was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mg mL−1 (17.7 mM). The running buffer for protein immobilization contained 1× PBS and 0.005% Tween 20, and that for compound analysis contained 1× PBS, 0.005% Tween 20, and 5% DMSO. The flow rates were 10 μL/min for protein immobilization and 30 μL/min for compound analysis. Proteins were immobilized on flow cell 2 of different channels of a series S CM5 sensor chip via an amine-coupling method to 5,000 to 6,000 response units (RU). Flow cell 1 of the corresponding channels of the sensor chip was left blank as a control. Compound A378-0 was injected with a concentration series of 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM, with one intermediate concentration injected as a duplicate. Both the association and dissociation periods were set to 60 s, and no regeneration was needed. Data were analyzed with Biacore Insight Evaluation software, and values from the first 5 s after the start of the injection were used to calculate the dissociation equilibrium constant (DK) by fitting to a 1:1 steady-state affinity model.EGRYRHLVALAT) of Swi6-1 was used as the substrate. The reaction buffer contained 40 mM Tris-HCl (pH 7.5), 20 mM MgCl2, and 0.1 mg mL−1 bovine serum albumin (BSA). Each sample contained 0.2 mg mL−1 protein, 0.1 mM ATP, 0.4 mM Swi6-1, and 0 to 25 μM compound A378-0, with a total volume of 50 μL and a final DMSO concentration of 2%.The kinase activity of Mps1 and Mpk1 was determined with the ADP-Glo kinase assay kit . A peptide . Later, a kinase detection reagent was added, and the samples were mixed gently and incubated for another 40 min to convert ADP into ATP. The newly synthesized ATP can be detected using a luciferase reaction, and being measured with a microplate reader  with absorbance at 620 nm. Each sample contained at least three replicates. The amount of newly synthesized ADP indicates the kinase activity of the tested protein.−1 and then mixed with A378-0 at a molar ratio of 1:1. The protein-compound mixture was incubated on ice for 1 h and then screened for crystals. Crystallization was performed using the sitting-drop diffusion method with the Oryx4 instrument (Douglas) at 18°C. Reservoir buffer was prepared with optimization of the apo-Mps1 crystallizing conditions (PDB accession number 5Z33). The total volume of each drop was 0.5 μL, and the volume ratio of protein to reservoir buffer was 3:7, 5:5, or 7:3. Crystals for data collection were obtained in a reservoir solution containing 20% to 30% Tacsimate, 0.05 M sodium cacodylate trihydrate (pH 6.5 to 7.5), and 0.1 mM spermine. For the collection of X-ray diffraction data, crystals were flash-frozen in liquid nitrogen and protected with reservoir solution supplemented with 20% glycerol.Mps1 protein in storage buffer was concentrated to 8.5 to 9.5 mg mL5Z33) using Phaser MR of the CCP4 software suite (Diffraction data were collected at Shanghai Synchrotron Radiation Facility Beamline 19U1 (SSRF BL19U1). Data were indexed, integrated, and scaled with HKL-2000 . The phare suite  and thenre suite  and COOTre suite . Data prre suite .M. oryzae, we carried out appressorium development, appressorium penetration, and infection assays. M. oryzae strain P131, barley cultivar E9, and rice cultivar Nipponbare were used in these experiments. P131 was maintained on oatmeal tomato agar (OTA) plates  plates . The con) plates . Compoun5 to 3 × 105 conidia mL−1) with or without A378-0 was then placed onto the glass coverslips. Dishes were incubated in a dark and humid chamber at 28°C for 12 h. Appressorium formation was observed and appressoria were counted with a Nikon microscope.For the appressorium development assay, 20-mm by 20-mm borosilicate glass coverslips were placed into a 9-cm petri dish, and 5 μL of the conidial suspension (2 × 105 to 3 × 105 conidia mL−1) with or without A378-0 and incubated in a dark and humid chamber at 28°C for 36 h. Appressorium penetration was observed with a Nikon microscope.For the appressorium penetration assay, the back sides of 1-week-old leaves of barley were spot inoculated with 5 μL of the conidial suspension (2 × 104 to 5 × 104 conidia mL−1) with or without A378-0 at different concentrations, incubated in a dark and humid chamber at 28°C for 36 h, transferred to an illuminated and humid chamber at 28°C, and incubated for another 3 to 4 days. Lesions were observed and recorded.For the infection assay, 8-day-old leaves of barley or 3-week-old leaves of rice were used. Before inoculation, the rice leaves were wounded mildly with a needle, while the leaves of barley needed no such pretreatment. The leaves were spot inoculated with 5 μL of the conidial suspension (3 × 108H59.The accession number for the Mps1/A378-0 complex structure in the PDB is


Genomic three-dimensional (3D) spatial organization plays a key role in shaping gene expression and associated chromatin modification, and it is highly sensitive to environmental stress conditions. In microalgae, exposure to nitrogen stress can drive lipid accumulation, yet the associated functional alterations in the spatial organization of the microalgal genome have yet to be effectively characterized.Nannochloropsis oceanica in response to nitrogen deprivation (ND). These analyses revealed that ND resulted in various changes in chromosomal organization, including A/B compartment transitions, topologically associating domain (TAD) shifts, and the disruption of short-range interactions. Significantly higher levels of gene expression were evident in A compartments and TAD boundary regions relative to B compartments and TAD interior regions, consistent with observed histone modification enrichment in these areas. ND-induced differentially expressed genes (DEGs) were notably enriched in altered TAD-associated regions and regions exhibiting differential genomic contact. These DEGs were subjected to Gene Ontology (GO) term analyses that indicated they were enriched in the ‘fatty acid metabolism’, ‘response to stress’, ‘carbon fixation’ and ‘photosynthesis’ functional categories, in line with the ND treatment conditions used to conduct this study. These data indicate that Nannochloropsis cells exhibit a clear association between chromatin organization and transcriptional activity under nitrogen stress conditions. Pronounced and extensive histone modifications were evident in response to ND. Observed changes in chromatin architecture were linked to shifts in histone modifications and gene expression.Accordingly, the present study employed RNA-seq, Hi-C, and ChIP-seq approaches to explore the relationship between 3D chromosomal architecture and gene expression during lipid accumulation in the marine microalga Overall, the reprogramming of many lipid metabolism-associated genes was evident under nitrogen stress conditions with respect to both histone modifications and chromosomal organization. Together these results revealed that higher-order chromatin architecture represents a new layer that can guide efforts to understand the transcriptional regulation of lipid metabolism in nitrogen-deprived microalgae.The online version contains supplementary material available at 10.1186/s13068-023-02378-0. Chlamydomonas reinhardtii . Signals were excluded from such transformation into Z-scores if they exhibited a count of 0. Z-scores were used as a means of correcting for small variations in the overall decay with genomic distance that may arise among samples.Overall chromatin interaction decay with distance was evaluated with a modified LOWESS method  as in prior reports 30. This approach pools genome-wide data and calculates weighted average and weighted standard deviation values for all genomic distances. Interaction data were transformed into C-score signals were divided into an active A compartment consisting of gene-dense regions of euchromatin and an inactive B compartment consisting of regions of gene-poor heterochromatin.In the 40-kb bin-based single chromosome contact heatmaps, a plaid-like pattern is visible, with the alternating regions of high- and low-frequency interactions corresponding to compartments A and B. These compartments were identified at a 100-kb resolution with the CscoreTool (v 1.1) software . GenomicTADs are contiguous genomic regions with a high degree of self-association and that have distinct boundaries separating them from nearby genomic regions. TAD locations can be defined when binning interaction data at 20 kb. For this study, TAD boundaries in individual samples were assessed with an insulation score algorithm   q-values. Interactions with a contact count > 2, a p-value < 0.01, and a q-value < 0.01 were considered significant.Intra- and inter-chromosomal interactions for each sample were assessed based on the contact between 10-kb bins using the Fit-Hi-C software (v2.0.8) with the settings: -L 10,000 -p2) . This apN. oceanica (1–2 million cells mL−1) from an 800 mL culture were fixed at room temperature using 1% formaldehyde (Sigma) under vacuum for 30 min, after which they were treated with 0.15 M glycine under vacuum for 10 min at room temperature. Nuclei were then isolated as reported previously, with each round of ChIP being performed with the nuclei isolated from fixed material [2, 10 µM ZnSO4, 1% Triton X-100, 0.05% SDS) followed by incubation overnight with anti-H3K36me2 , anti-H3K27Ac (Abcam ab4729), or anti-Kcr  at 4 °C. Then, samples were mixed with 50 µL of Protein A/G magnetic beads (Millipore) followed by a 2 h incubation at 4 °C. Beads were subsequently washed (5 min/wash) three times using IP buffer, one time using IP buffer supplemented with NaCl (500 mM), and one time with LiCl buffer . Then, 200 µL of elution buffer  was incubated with chromatin-bound beads for 30 min at 65 °C, after which samples were treated for 6 h with Proteinase K at 65 °C. A Qiagen kit (Qiagen) was subsequently used to extract DNA from these samples, which were then subjected to end repair, A-tailing, adaptor ligation, and library amplification based on standard protocols (Illumina). An Illumina HiSeq 2500 instrument was then used to sequence (1 × 150 bp) the resultant library, with a slightly modified version of the nfcore/chipseq pipeline [https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/), followed by alignment with BWA [N. oceanica reference genome. Picard was used to mark duplicate mapped reads, which were analyzed with DeepTools [material . Brieflypipeline  being uswith BWA  to the NeepTools  and MACSeepTools . Peak caeepTools  was usedeepTools  package g for 5 min, snap-frozen with liquid nitrogen, and stored at − 80 °C for further analysis. Trizol  was used to extract total RNA from these samples, after which a NanoDrop-1000 instrument  was used to evaluate RNA sample concentrations and purity. Sera-mag Magnetic Oligo (dT) Beads  were used to purify poly-adenylated mRNAs from these samples, followed by incubation in an RNA Fragmentation Reagent  to generate 200–300 bp fragments based on provided directions. Agencourt RNA Clean beads  were then used to purify these fragmented RNA sequences, which were used for cDNA synthesis with the SuperScript Double-Stranded cDNA Synthesis Kit  using random hexamer primers. A NEBNext® mRNA Library Prep Reagent Set  was then used for strand-nonspecific library preparation, followed by 2 × 150 bp paired-end sequencing with an Illumina HiSeq2000 instrument. Raw data were deposited in the NCBI GEO database with the accession number GSE225079.Cells were centrifuged at 2500×https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/), with remaining high-quality reads then being aligned to the N. Oceanica IMET1 genome (http://nandesyn.single-cell.cn/download) using STAR [P-value ≤ 1 when comparing control and ND conditions, with CPM values for at least two samples ≥ 1.The nfcore/rnaseq pipeline was used for analyses of RNA-seq results . Brieflying STAR , with Piing STAR  and Striing STAR . Gene exing STAR , with noing STAR . SignifiThe data were analyzed via one-way ANOVA with a least significant difference test.Additional file 1: Figure S1. Experimental design and physiological measurement under N repletion and deprivation. (A) Experimental design for Hi-C, ChIP-seq and mRNA-seq data collection. (B) OD750, photosynthetic parameter (Fv/Fm) and photosynthetic oxygen evolution (POE) under nitrogen repletion and deprivation. Figure S2. Depth in different resolutions. The matrix resolution of a Hi-C map was defined as the smallest locus size such that 80% of loci have at least 1000 contacts. The resolution of > 2 kb is suitable for following analysis. Figure S3. Minus heatmap matrix of each chromosome in response to nitrogen deprivation between C0 and N1. At 2kb resolution, subtraction heatmap matrix of each chromosome was constructed between C0 and N1. Figure S4. Minus heatmap matrix of each chromsome in response to nitrogen deprivation between C0 and N2. At 2kb resolution, subtraction heatmap matrix of each chromosome was constructed between C0 and N2. Figure S5. Dekay curve of interaction frequencies against increasing genomic distance for each chromosome in N. oceanica. The genomic bin size is 100kb. Figure S6. Gene density and GC content in the compartment A/B under C0 vs. N1 and C0 vs. N2. (A) GC content in the region of compartment A and B under C0, N1 and N2. (B) Gene density in the region of compartment A and B under C0, N1 and N2. (C) Comparison of compartment length in the region of compartment A and B under C0, N1 and N2. Figure S7. Genome eigenvector analysis of compartment A/B in chromosome 23, 26 and 30. Segregation of local A/B compartments using eigenvector under C0, N1 and N2 for chromosome 23, 26 and 30 in N. oceanica. Blue and dark-red represented compartment A and B, respectively. Figure S8. Gene density and GC content in the transition region of compartment A/B under C0 vs. N1 and C0 vs. N2. A GC content in the compartment A/B switching region under C0 vs. N1 and C0 vs. N2. B Gene density in the compartment A/B switching region under C0 vs. N1 and C0 vs. N2. Figure S9. Gene density and GC content in the region of TAD border and inter under C0 vs. N1 and C0 vs. N2. (A) GC content in the region of TAD border and inter under C0, N1 and N2. (B) Gene density in the region of TAD border and inter under C0, N1 and N2. (C) The top five motifs in the region of TAD border C0, N1 and N2. Figure S10. Venn diagram of TAD border between C0 and N1, or C0 and N2, or N1 and N2. The number of TAD border were compared between C0 and N1, or C0 and N2, or N1 and N2. Figure S11. Pearson correlation relationships of mRNA-Seq libraries. (A) Pearson correlation relationships of six mRNA-Seq libraries. (B) PCA analysis of six sequencing samples. Figure S12. Comparsion of the gene expression in TAD transition regions under C0 vs. N1 and C0 vs. N2. (A) and (B) showed gene expression level in TAD transition regions under C0 vs. N1 and C0 vs. N2, respectively. Figure S13. KEGG functional enrichment of up-regulated genes in compartment A/B and TAD under C0 vs. N1 and C0 vs. N2. (A) and (B) KEGG functional enrichment of up-regulated genes in compartment A/B under C0 vs. N1 and C0 vs. N2. (C) and (D) KEGG functional enrichment of up-regulated genes in the TAD bonder under C0 vs. N1 and C0 vs. N2. Figure S14. Correlation coefficients of ChIP-seq libraries. Heatmap showed correlation of different samples. Figure S15. Distribution of enrichment analysis in histone modifications. Histone modification enrichment profile across genes were showed. Figure S16. Pattern of histone modifications over 6kb regions surrounding the TSS for H3K27ac and H3K36me2.Additional file 2: Table S1. Statistics of Hi-C sequencing data. Table S2. Percent of the genomic regions in compartment A/B. Table S3. Statistics of stable and switch compartment A/B. Table S4. Statistics of mRNA sequencing data. Table S5. Statistics of ChIP sequencing data. Table S6. Static of total peaks in three modifications . Table S7. Statistics of overlap peaks for H3K27ac, H3K36me2 and Kcr between C0 and N1, or C0 and N2.


Rapid and high-throughput screening of antiviral clustered regularly interspaced short palindromic repeat (CRISPR) RNAs (crRNAs) is urgently required for the CRISPR-Cas13a antiviral system. Based on the same principle, we established an efficient screening platform for antiviral crRNA through CRISPR-Cas13a nucleic acid detection.In this study, crRNAs targeting PA, PB1, NP, and PB2 of the influenza A virus (H1N1) were screened using CRISPR-Cas13a nucleic acid detection, and their antiviral effects were confirmed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The RNA secondary structures were predicted by bioinformatics methods.The results showed that crRNAs screened by CRISPR-Cas13a nucleic acid detection could effectively inhibit viral RNA in mammalian cells. Besides, we found that this platform for antiviral crRNA screening was more accurate than RNA secondary structure prediction. In addition, we validated the feasibility of the platform by screening crRNAs targeting NS of the influenza A virus (H1N1).This study provides a new approach for screening antiviral crRNAs and contributes to the rapid advancement of the CRISPR-Cas13a antiviral system. The influenza virus is a negative-sense single-stranded RNA virus that belongs to the Orthomyxoviridae family and causes respiratory diseases . The worThe clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) system is a defense mechanism that protects against viruses and plasmids in bacteria and archaea . Previouin vitro, rapid screening of efficient antiviral targets is possible. In this study, an efficient screening technology for antivirus crRNA targets was established based on CRISPR-Cas13 nucleic acid detection using the influenza virus (H1N1) as an example. The screened crRNAs have high anti-influenza virus activity. It provides a new strategy for programmable antiviral target screening based on the CRISPR-Cas13a system.Similar to intracellular antiviruses, the CRISPR-Cas13 system has also been used for nucleic acid detection , 16, 17.An IAV (H1N1) subtype-specific crRNA was designed Table 1.2O, 7.5μL. The reaction procedure is as follows: 37°C, 15min, 1cycle; 85°C, 5min, 1cycle; 95°C, 30s, 95°C, 10s, 55°C, 30 s, 40cycle; 72°C, 30s, 1cycle; 72°C, 10min,1cycle; 4°C, ∞. For CRISPR detection, The reaction system was as follows: 1.6 IU/μL RNase inhibitor (NEB), 20 mM N-2-hydroxyethyl piperazine-N-2-ethane sulfonic acid , 25 nM Cas13a (Genscript), 2 μM crRNA, 2.5 mM ribonucleoside triphosphates (NEB), 2 nM reporter RNA (Invitrogen), 1 IU/μL T7 RNA polymerase (NEB), 10 mM MgCl2, and 5 μL of the RT-PCR amplified product. The reaction was incubated and detected for 60 min at 37°C using the fluorescence quantitative PCR instrument MasterCycler RealPlex4 (Eppendorf).The RT-PCR amplified template was the RNA of the influenza A PR8 strain. The primers were listed in A549 and Madin-Darby canine kidney (MDCK) cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin and streptomycin, and 100 mg/mL L-glutamine. A/Puerto Rico/8/1934(H1N1) (provided by the Center for Disease Control and Prevention of the Chinese People’s Liberation Army) viral culture supernatant was harvested in minimal Eagle medium (MEM) from MDCK cells, filtered through a 0.2 mm filter, titrated by a plaque test on MDCK cells, and stored at -80°C.2 for 24 h. Cells were harvested to extract RNA and viral RNA was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Infectious viral particles were verified by median tissue culture infectious dose (TCID50), and plaque assays.During virus adsorption, crRNA/Cas13a complexes were prepared according to optimized conditions: 500 ng Cas13a protein, 125 ng crRNA, 1 μL Reagent PLUS, and 25 μL Opti-MEM in tube 1, and 1.5 μL Lipofectamine CRISPR MAX reagent (Thermo) and 25 μL Opti-MEM in tube 2. The components were mixed thoroughly and incubated for 5 min at room temperature, after which those of both tubes were mixed and incubated at RT for 25 min. The complexes containing crRNA-Cas13a, Cas13a alone, or the transfection reagent alone were added to PR8-infected cells and incubated for 4 h. Finally, 0.5 mL of DMEM containing 1% bovine serum albumin (BSA), 1% penicillin and streptomycin, glutamine, and 1 mg/mL L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) were added to each well and incubated at 37°C and 5% COViral RNA was extracted from cell particles using a QIAamp Viral RNA Mini kit (Qiagen), after which viral RNA was quantified using a one-step TB Green PrimeScript RT-PCR kit (TAKARA). A universal primer pair for detecting the Matrix protein (M) segment of the influenza A virus was used, the forward primer was 5’-TTCTAACCGAGGTCGAAACG-3’, the reverse primer was 5’-ACAAAGCGTCTACGCTGCAG-3’. β-actin  was used as an internal control. The reactions were performed as follows: reverse transcription at 42°C for 5 min, denaturation at 95°C for 10 s, and 40 cycles of amplification at 95°C for 5 s and 60°C for 30 s.2 for 90 min to allow virus adhesion, dissolved with 1.5% methylcellulose, mixed with 2× DMEM in an equal proportion, and covered with 1% BSA and 2 mg/mL TPCK pancreatic enzyme. The cells were incubated at 37°C and 5% CO2 for 3 days and fixed with 4% paraformaldehyde at room temperature for 30 min. After washing with water, 2% crystal violet dye was added to the cells, then incubated at room temperature for 30 mins. After washing with water, plaques were counted, and the viral titer was expressed as a plaque-forming unit (PFU)/mL.Different experimental samples were diluted 10 times and then added to MDCK cells in 12-well plates. Then they were incubated at 37°C and 5% CO2 for 90 min to allow the virus adhesion. After discarding the diseased venom, DMEM containing 1% BSA and 2 mg/mL TPCK trypsin was added to each well. The cells were incubated at 37°C and 5% CO2 for 3 days, and cytopathy was observed daily. The viral titer was calculated after the cells stopped exhibiting pathological changes, and the viral titer was represented as IogTCID50.After 10-fold gradient dilution, different experimental samples were added to MDCK cells in 96-well plates and incubated at 37°C and 5% COt-test using GraphPad Prism 8.0.2 software. Data are presented as the mean ± standard error of the mean. The levels of significance were indicated as follows: n.s., no significant difference; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.Statistical significance was determined by unpaired two-tailed Student’s via sequence alignment. Six crRNAs were designed for each segment , which is composed of PA, PB1, and PB2 proteins, binds to and enters the nucleus to initiate replication and transcription, forming mRNA for the production of other viral proteins. PA, PB1, and PB2 are critical for IAV replication and transcription , 27. NP The secondary structure of crRNA could affect the antiviral effect of the CRISPR-Cas13 system. The binding of crRNA to target RNA can affect the cleavage activity of the CRISPR system . MeanwhiIn conclusion, this study is the first to combine the CRISPR-Cas13a nucleic acid detection and antiviral technologies to establish a high-efficiency anti-influenza virus crRNA target screening technology, and it demonstrated the feasibility of this technology through further research. The platform allows for efficient, rapid, high-throughput, and low-cost screening against influenza virus crRNA targets, shortens the screening cycle of CRISPR antiviral targets, and provides a powerful tool for promoting the application and development of programmable antiviral technologies based on the CRISPR-Cas13 system.The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding authors.YSS, HL, YZ, and LY conceived the experiments. YZ, WY, XD, and MN performed the experiments. LY performed the RNA structure prediction. YZ and YH conducted statistical analysis. LY, YZ, and WY prepared the tables and figures. LY, HL, YZ, and YJS prepared the manuscript, LY and HL revised the manuscript. All authors have reviewed and approved the final manuscript.