Autophagy is a conserved recycling process in cells. However, the effects of autophagy on the remission and treatment response of acute myeloid leukemia (AML) patients have not been clarified.MAP1LC3B, ATG5, ATG10, RB1CC1, and AMBRA1 genes was assessed in 32 newly diagnosed AML patients, 18 complete remission (CR) patients, and seven relapsed patients, as well as 15 controls, by real‐time polymerase chain reaction (PCR).The expression of p < 0.0001). The MAP1LC3B, ATG5, ATG10, RB1CC1, and AMBRA1 gene expression significantly reduced in CR patients compared to newly diagnosed AML patients . The AMBRA1 gene expression was significantly higher in the relapsed cases as compared to both newly diagnosed (p = 0.01) and CR patients (p = 0.03). Moreover, a significant positive correlation was observed between the expression of MAP1LC3B , ATG5 , and ATG10  genes and white blood cell (WBC) count in patients at diagnosis.The expression of all five genes was significantly higher in the newly diagnosed AML patients as compared to the controls (MAP1LC3B, ATG5, ATG10, RB1CC1, and AMBRA1 genes can be examined to follow‐up the remission of AML and the patient's response to treatment.The expression of  Autophagy is known as an effective pathway in biological processes, but their unknowns are still secret in leukemia and response to treatment. The autophagy‐related genes might be changed and useful to remission follow‐up. In this study, we try to introduce the impact of autophagy‐related genes in pathogenesis, treatment response, and remission of acute myeloid leukemia. These patients were classified according to the treatment regimen (AML‐M3 and non‐M3 AML) at the Hematology Department of Namazi Hospital, affiliated to Shiraz University of Medical Sciences, Shiraz, Iran.The patients received the standard‐of‐care regimens , and after the induction phase, 18 patients attained CR. Patients who expired were excluded from the study. We also included seven AML patients in the relapse phase. Moreover, we recruited 15 healthy individuals without cancer or hematological diseases as the control group; they were matched with AML patients in terms of age and gender.2.2Total RNA was extracted from whole blood samples using TRIzol , according to the manufacturer's instructions. Subsequently, the extracted RNA concentrations were measured by a NanoDrop instrument . Next, 0.4 μg of each RNA was reverse‐transcribed into cDNA to a final volume of 10 μl, according to the instructions of the PrimeScript First‐Strand cDNA Synthesis Kit .2.3MAP1LC3B (GenBank Ref Seq no: NM_022818.5), ATG5 (GenBank Ref Seq no: NM_004849.4), ATG10 (GenBank Ref Seq no: NM_031482.5), RB1CC1 (GenBank Ref Seq no: NM_014781.5), and AMBRA1 (GenBank Ref Seq no: NM_001267782.2) genes, using Qiagen real‐time PCR cycler . Beta‐actin was used as an endogenous control for normalization of mRNA expression between different samples. For each RT‐PCR reaction, 1 μl of cDNA, 10 μl of SYBR Green PCR Master Mix , 8.2 μl of nuclease‐free water, 0.4 μl of forward primer, and 0.4 μl of reverse primer in a final volume of 20 μl were used. The fold changes in MAP1LC3B, ATG5, ATG10, RB1CC1, and AMBRA1 gene expression were analyzed using the 2−∆∆CT method.AlleleID 7.0 and Gene Runner were used to design the sequences of specific primers. A qRT‐PCR was performed to evaluate the expression of 2.4t‐test, and unpaired t‐test were performed to compare the gene expression between each two groups. Fold changes were calculated using the 2−∆∆CT method. Moreover, the correlations between gene expression and the clinical characteristics of patients were analyzed using Pearson's correlation coefficient (r) test. p‐value <0.05 was considered to be statistically significant in all tests.Statistical analysis was performed using GraphPad Prism 8.0.2  and SPSS version 25.0 . Mann–Whitney test, Wilcoxon test, paired 33.1The clinical characteristics of 32 newly diagnosed AML patients, 18 AML patients in the CR phase, and seven patients in the relapse phase, as well as 15 healthy controls are presented in Table 3.2MAP1LC3B, ATG5, ATG10, RB1CC1, and AMBRA1 genes was evaluated in 32 newly diagnosed AML (new case) patients and 15 healthy controls. The results showed that the expression of MAP1LC3B, ATG5, ATG10, RB1CC1, and AMBRA1 genes was significantly upregulated in AML patients as compared to the control group  (p < 0.0001). The details of these results are shown in Figure The mRNA expression of 3.3MAP1LC3B, ATG5, ATG10, RB1CC1, and AMBRA1 genes was investigated in 18 newly diagnosed AML patients and 18 AML‐CR patients in the same cases. The results showed that the expression of MAP1LC3B, ATG5, ATG10, RB1CC1, and AMBRA1 genes was significantly downregulated in CR patients as compared to newly diagnosed AML patients  . The relative expression of these genes in CR patients compared to newly diagnosed AML patients is shown in Figure The expression of MAP1LC3B, ATG5, ATG10, RB1CC1, and AMBRA1 genes was assessed in seven AML patients in the relapse phase and compared with that of 32 newly diagnosed AML patients. There was no significant difference in the expression of MAP1LC3B, ATG5, ATG10, and RB1CC1 genes between relapsed and newly diagnosed cases (p > 0.05). The AMBRA1 gene expression significantly increased in patients with relapse as compared to newly diagnosed AML patients (2.1 folds) (p = 0.01); the details are presented in Figure Moreover, the expression of MAP1LC3B, ATG5, ATG10, RB1CC1, and AMBRA1 genes in seven relapsed cases versus 18 CR cases. The results showed that the expression of MAP1LC3B, ATG5, ATG10, and RB1CC1 genes was not significantly different between the relapse and CR groups (p > 0.05). However, the expression of AMBRA1 gene was significantly higher in relapsed patients as compared to CR patients (2.8‐folds) (p = 0.03); this comparison is presented in Figure Moreover, to determine the role of autophagy‐related genes in the chemo‐resistance of AML patients, we compared the expression of 3.4MAP1LC3B, ATG5, ATG10, RB1CC1, and AMBRA1 genes and age, gender, hemoglobin (Hb) level, white blood cell (WBC) count, platelet count, bone marrow blast percentage, and subtype at diagnosis and in the CR and relapse phases were investigated. There was no significant association between the expression of these genes and age, gender, Hb level, platelet count, bone marrow percentage, and subtype of patients at diagnosis.The correlations between the expression of MAP1LC3B , ATG5 , and ATG10  genes and WBC count. Therefore, upregulation of MAP1LC3B, ATG5, and ATG10 genes was associated with a high WBC count at diagnosis. In CR patients, no significant correlation was observed between the expression of MAP1LC3B, ATG5, ATG10, RB1CC1, and AMBRA1 genes and the patients' clinical characteristics (p > 0.05). Also, the results did not indicate a significant correlation between the expression of these genes and the clinical characteristics of relapsed patients (p > 0.05).However, a significant positive correlation was found between the expression of 3.5MAP1LC3B, ATG5, ATG10, RB1CC1, and AMBRA1 genes and CD34+ marker was examined in this study. According to the results, there was no significant correlation between the expression of MAP1LC3B, ATG5, ATG10, RB1CC1, and AMBRA1 genes and CD34+ marker (p > 0.05); the results are presented in Figure The relationship between the expression of 3.6MAP1LC3B, ATG5, ATG10, RB1CC1, and AMBRA1 genes. The results showed a significant positive correlation between MAP1LC3B, ATG5, ATG10, and RB1CC1 genes (p < 0.05). Besides, the expression of AMBRA1 gene had a significant positive correlation with ATG5 and RB1CC1 genes (p < 0.05).A statistical analysis was performed to evaluate the correlations between 4RB1CC1 gene. This protein, along with ULK1, ATG13, and ATG101, forms the ULK complex, which activates autophagy under various conditions, such as starvation and treatment with rapamycin. Autophagosome nucleation is triggered by activation of class III phosphatidylinositol 3‐kinase (PI3KC3) complex. PIK3C3, Beclin1, AMBRA1, and P150 form the core of the PI3KC3 complex. Generally, the vesicle elongation and maturation phase depends on two ubiquitin‐like conjugation reactions: ATG5‐ATG12 and LC3‐PE conjugation. ATG7 (an E1‐like enzyme), as well as ATG10 and ATG3 (E2‐like enzymes), is involved in these conjugation reactions. During the vesicle elongation and maturation phase, ATG4 cleaves LC3 to LC3I. Then, LC3I is converted into LC3II, which remains on the autophagosomes membrane and is used as an autophagy marker . FIP200 is an essential protein for autophagy induction, encoded by the Autophagy may affect the initiation and progression of leukemias. Leukemic cells can utilize autophagy to provide their metabolic needs for proliferation and survival. Also, autophagy activation can protect leukemic cells against oxidative stress; therefore, it can promote leukemogenesis, as well as drug resistance following treatment    as compared to newly diagnosed AML patients   could enhance cytarabine cytotoxicity against the HL‐60 cell line , ATG5 , and ATG10  genes was related to a high WBC count at diagnosis. Based on our results and studies on the role of autophagy in myeloid cell proliferation, differentiation, and apoptosis   in AML and myelodysplastic syndrome (MDS) patients List, . SeveralWe recommend that future studies focus on the correlation of autophagy‐related genes with the prognosis of AML patients. We could not evaluate the levels of LC3, ATG5, ATG10, RB1CC1, and AMBRA1 proteins because of some limitations. Therefore, future studies need to measure these proteins using the western blot technique. Also, further studies are needed to investigate the relationship between autophagy‐related genes and minimal residual disease (MRD).5MAP1LC3B, ATG5, ATG10, RB1CC1, and AMBRA1, could be related to the pathogenesis of AML and the patients' response to treatment. Therefore, these genes may be suggested as therapeutic biomarkers to follow‐up the CR status. Also, inhibition of autophagy‐related genes might be beneficial in inducing remission, preventing relapse, and also, improving the treatment outcomes of AML patients.The present results showed that the key autophagy‐related genes, including The authors declare no conflict of interest.Parisa Tandel designed the study, performed the research, analyzed the data, and wrote the manuscript. Gholamhossein Tamaddon designed the study and was responsible for data management and analysis. Reza Ranjbaran revised the article critically and contributed to data analysis. Eqbal Ebrahimi performed the research and analyzed the data. Mani Ramzi and Alireza Rezvani revised the article critically.The local Ethics Committee of Shiraz University of Medical Sciences approved this study (IR.SUMS.REC.1397.085), and the written informed consent was obtained from all participants.


OTUs) that were >0.1% abundant (average) in the donor sample, 137 were detected in the treated group (75 were >0.1% abundant (average)) while only 88 were detected in the control group (29 were >0.1% abundant (average)). Our data therefore suggests that stable transplantation of the cecal microbiota between lines is achievable using the methods described in this paper.The cecal microbiota plays numerous roles in chicken health and nutrition. Where such microbiota differs between lines exhibiting distinct phenotypes, microbiota transplantation offers scope to dissect the role of gut microbial communities in those traits. However, the composition and stability of transplants over time is relatively ill-defined and varying levels of success have been reported. In this study, we transplanted cecal contents from adult Roslin broilers into chicks from a different broiler line. Within <12 h posthatch, Ross 308 chicks received an oral gavage of cecal contents (n = 26) or a PBS control (n = 24). Cecal contents samples were collected postmortem from birds on d 1, 2, 3, 4, and 7 posthatch. DNA was extracted from these samples and the transplant inoculum and the V4 region of the 16S rRNA gene was amplified and sequenced. The cecal microbiota of chickens receiving the microbiota transplant was significantly different in composition and significantly richer and more diverse, in comparison to control birds. At the final timepoint (d 7), of the 150 Operational Taxonomic Units ( The ceca harbor the greatest concentration of microbes in the chicken gastrointestinal tract. These microbial communities are commonly referred to as the cecal microbiota. Despite advances in knowledge from metabarcoding, metagenomic and culturing studies, there are still many questions left to answer about the impact of the cecal microbiota on factors such as feed conversion efficiency and resistance to colonization by pathogens. Recent metagenomic studies have highlighted that there remain many uncultured and unstudied microorganisms in the chicken ceca , which mStudies have shown varying success when attempting to transplant gastrointestinal microbiota in chickens, potentially due to the variability of the methods used. Storage of cecal contents or feces prior to administration, or the exposure of these samples to oxygen, may impact the viability of some of the bacteria they contain. It would also be expected that the type of inoculum used will affect the likelihood of a successful transplantation.. demonstrated that administration of microbial transplants to young chicks induced long-term effects on their microbiota composition, though the recipient microbiota did not exactly reflect that of the donor . We used freshly collected cecal contents as an inoculum, to provide as accurate a representation of the cecal microbiota as possible. Cecal contents were transplanted within 30 min of collection. We also administered the cecal inoculum within a few h of hatch to decrease the likelihood of chicks being exposed to environmental bacteria prior to transplantation. We then characterized the development of the cecal microbiota over the first 7 d of life relative to the donor inoculum and control birds, as this is the time period identified in previous studies as being most impacted by transplantation.Animal procedures were performed in compliance with the Animals (Scientific Procedures) Act of 1986 and approved by the Animal Welfare and Ethical Review Board of The Roslin Institute. Donor birds weighing >3 kg were killed by captive bolt and recipient birds weighing <3 kg were killed by cervical dislocation in accordance with Schedule 1 of the Act. All chickens were hatched/housed at the National Avian Research Facility at The Roslin Institute, a Home Office accredited breeding establishment.Contents were collected postmortem from the 2 ceca of three 40-we"?>k-old Roslin broilers . These were mixed and diluted in phosphate-buffered saline (PBS) at a 1:6 w:w dilution . Within Cecal contents were taken postmortem from chicks on d 1 [5 control chicks  and 5 treated chicks ]; d 2 [5 control chicks  and 5 treated chicks ]; d 3 [5 control chicks  and 5 treated chicks ]; d 4 [5 control chicks  and 5 treated chicks ]; and d 7 [4 control chicks  and 6 treated chicks ]. To collect cecal contents, the abdomens of the birds were sprayed with 70% ethanol and the ceca were exposed using sterile scissors. The ceca were removed using sterile scissors at the junctions between the ceca and the large intestine. Luminal contents from both ceca were manually expressed into the same tube, then mixed and stored at -20°C until DNA extraction.DNA extraction reagent only controls (containing no sample) were produced for each DNA extraction batch, to act as negative controls . A mock NMDS) graphs using Bray-Curtis dissimilarity values. The Vegan package “adonis” function was used to perform PERMANOVA analyses. Richness (Inverse Simpsons) and diversity (Chao 1) indices were calculated and compared between groups using the Kruskal-Wallis test. ANCOM . Graphs were produced using the gpplot2 (v.3.2.1) and Venn Diagram (v.1.7.0) packages. The Vegan (v.2.5-6.) package was used to construct Nonmetric Multidimensional Scaling (t. ANCOM  (v.2.1) https://doi.org/10.7488/ds/3129.The paired-read fastq files generated and analyzed during the study are available in the European Nucleotide Archive under project PRJEB46338. OTU tables can be found at Propionibacteriaceae (Cutibacterium acnes). The most abundant chicken microbiota phyla (Bacteroidota and Firmicutes). 10,416 OTUs were identified in our samples. All phyla present in the original mock community were present in the sequenced samples after subsampling (Supplementary Table 1), although members of the Actinobacteria were under-represented. All expected families were present, except for rmicutes) were welClostridium_sensu_stricto_1: 41 ± 28%), and OTU1 (Escherichia-Shigella: 29 ± 34%) (Escherichia bloom in 2 different chicken lines (Clostridium_sensu_stricto_1 bloom in Ross 308 birds (Lachnospiraceae_unclassified: 20 ± 11%) and OTU4 (Enterobacteriaceae_unclassified: 18 ± 12%) greatly increased in abundance. OTUs 1, 2 and 4 remained the most abundant OTUs at d 3 and 4. At d 7 the most abundant OTUs were OTU9 (Bifidobacterium: 8.3 ± 6.0%), OTU2 (Lachnospiraceae_unclassified: 5.4 ± 4.7%), OTU13 (Lachnospiraceae_unclassified: 5.4 ± 0.20%), OTU12 (Lactobacillus: 4.7 ± 1.7%), and OTU46 (Ruminococcaceae-Incertae_Sedis: 4.1 ± 0.62%). This progression over the first week, from a community dominated by Clostridium_sensu_stricto_1 to a more diverse microbiota including members of the Lachnospiraceae and Lactobacillaceae reflects our previous findings in this chicken line  . At d 1 en lines  and a Cl08 birds . At d 2,ken line .Figure 1Fusobacterium: 18 ± 3.1%), OTU1 (Escherichia-Shigella: 11 ± 2.2%), and OTU14 (Bacteroides: 7.1 ± 4.5%). Interestingly, these OTUs were far less abundant in the donor sample . These OTUs may represent bacterial species from the donor or environment that is able to colonize the bird more quickly than other species. The most abundant OTUs at d 2 were OTU6 , OTU7 (Phascolarctobacterium: 6.0 ± 1.0%), OTU14 (Bacteroides: 4.3 ± 3.8%), OTU8 (Fusobacterium: 4.2 ± 2.0%) and OTU18 . Several of these OTUs were found in similar abundances in the donor sample: OTU6 (3.4 ± 0.14%), OTU7 (2.6 ± 0.4%) and OTU18 (2.3 ± 0.088%). At d 7, the most abundant OTUs were OTU10 (Anaerobiospirillum: 6.6 ± 1.8%), OTU16 (Bacteroides: 5.2 ± 1.6%), OTU7 (Phascolarctobacterium: 5.2 ± 1.1%), OTU15 (Megamonas: 5.0 ± 2.1%) and OTU6 . In the donor sample several of these OTUs were present in similar proportions while others were less abundant: OTU10 (0.084 ± 0.016%), OTU16 (0.72 ± 0.087%), OTU7 (2.6 ± 0.43%), OTU15 (0.15 ± 0.03%), and OTU6 (3.4 ± 0.14%). Overall, a greater abundance of OTUs were shared between the 2 donor samples and the treatment samples than between the donor samples and the control group (Akkermansia: 7.6 ± 0.47%), OTU128 (UCG-005: 3.0 ± 0.30%), OTU56 (Rikenellaceae_RC9_gut_group: 3.0 ± 0.080%), and OTU133 . Chicken cecal contents separated differently by their compositions according to whether they received the cecal transplant or the PBS control (P = 1e-05), with the treated samples clustering more closely to the donor samples (P = 1e-05) (P = 1e-05). The donor sample contained on average 150 OTUs that were >0.1% abundant (average); at d 1, 136 of these OTUs were detected in the treated group [54 OTUs >0.1% abundant (average)] while 73 of these OTUs were detected in the control group [8 OTUs >0.1% abundant (average)]. At d 7, 137 of these OTUs were detected in the treated group [75 OTUs >0.1% abundant (average)] while 88 of these OTUs were detected in the control group [29 OTUs >0.1% abundant (average)]. This indicates that while some OTUs were not successfully transplanted, cecal content transplantation had a major impact on the microbiota, and on the development of the microbiota over time.In comparison, the cecal microbiota of birds that received microbial transplants was dominated by different bacterial OTUs. At d 1 the most abundant OTUs were OTU8  . The intThe most notable taxonomic difference between treated and untreated samples is the abundance of the phylum Bacteroidota, which is present at a high abundance in the donor sample (38 ± 1.4%). At d 1, this taxa was found at very low abundances in control samples (0.022 ± 0.0081%) but in high abundance in treated samples (28 ± 2.9%). By 7 d, the relative abundance of Bacteroidota in control samples had barely changed (0.020 ± 0.0039%), while in treated samples it had increased further (41 ± 4.4%). These results reflect those of Kubasova et al., who found that adult hens were efficient donors of Bacteroidetes when housed with young chicks .At d 1, 114 OTUs were found to be differentially abundant between the treatment and control group. The number of OTUs differentiating the 2 groups then sharply increased at d 2 (226 OTUs), reflecting the increase in diversity of microbial communities over time . At d 3 P = 8.346e-08) and diversity  than chickens that received control PBS. The diversity and richness of the microbiota increased over time for both groups, but did not reach the same levels as the donor communities by the end of the study period (The cecal microbiota of chickens that received a transplant of cecal contents had significantly higher richness (Kruskal-Wallis: y period . This isy period .In conclusion, we were able to successfully transplant the majority of the bacterial members of the cecal microbiota from 40-wk-old broilers to chicks from a different broiler line. We observed significant differences in composition and diversity at all-time intervals sampled, and transplantation clearly changed the trajectory of cecal microbiota development. This technique could be used in future studies to transplant the microbiota between broiler lines with desired phenotypes, to assess the impact of the microbiota on those phenotypes.


The highest ferromagnetic transition temperature  is found for low Mn and high Si contents, while the lowest is found for low Fe and Si contents  in the MnxFex2−Py1−Siy phase diagram. The largest hysteresis (91 K) was observed for a metal ratio close to Fe:Mn = 1:1 . Both Mn-rich with high Si and Fe-rich samples with low Si concentration were found to show low hysteresis (≤2 K). These compositions with a low hysteresis form promising candidate materials for thermomagnetic applications.The phase diagram of the magnetocaloric Mn The application or removal of an external magnetic field on a magnetic solid under adiabatic conditions triggers a temperature change that is known as the magnetocaloric effect (MCE) . In an i5Si2Ge2 alloys near ambient temperature [13-based alloys [2-type compounds have been considered as one of the most promising candidates for near-room temperature magnetic refrigeration and magnetic energy conversion applications due to their combination of a tuneable working temperature, low hysteresis, corrosion resistance, compositional stability and low material costs.The key to convert current thermomagnetic motors/generators into a cost-effective application is to optimise the magnetocaloric materials. Pecharsky and Gschneidner at Ames Lab discovered a giant magnetocaloric effect in Gdperature ,7,8. Sind alloys ,10,11, Md alloys ,15,16 and alloys ,18,19,202 compounds with varying Si-content have been thoroughly investigated for their giant MCE based on the earth-abundant and non-toxic elements [2 compounds while preserving a giant MCE [In the last decade, elements ,26,27,28iant MCE ,30,31,322 compounds and illustrated the composition dependence of the Curie temperature TC (K) and the thermal hysteresis ΔThys for the MnxFe2−xP1−ySiy compounds . It has been found that an increase in the Mn:Fe atomic ratio causes a reduction in both TC (K) and in thermal hysteresis ΔThys, while an increase in the P:Si atomic ratio leads to a decrease in TC and an increase in thermal hysteresis. This study focused on a limited region of the Fe-Mn-P-Si phase diagram. A revised FeMnPy1−Siy phase diagram was later presented by Höglin [2P-type (y < 0.15) structure and the hexagonal Fe2P-type (0.24 ≤ x ≤ 0.50) structure.Dung and co-workers  have cony Höglin , which cxFex2−Py1−Siy system is presented, with focus on the magnetocaloric active Fe2P-phase for a wide range of compositions (for Mn fraction of 0.3 < x < 2.0 and a Si fraction of 0.33 ≤ y ≤ 0.6). This extended phase diagram enables the search of suitable compositions for magnetic refrigeration and energy conversion applications. Furthermore, the presence of impurity phase(s) and the relationship between the heat of transformation of the magnetic transition and thermal hysteresis are discussed.In the present study, an extended phase diagram of the MnxFex2−Py1−Siy samples have been prepared by ball milling the starting materials Fe (99.9%), Mn (99.9%), red-P (99.7%) and Si (99.9%) powder. After 10 h of ball milling, the samples were pressed into tablets [Polycrystalline Mn tablets . The tabKα radiation. The lattice parameters and the impurity phases were obtained by a full Rietveld analysis of the X-ray diffraction data using the FULLPROF package [Powder diffraction patterns were collected in a PANalytical X-pert Pro diffractometer with Cu  package . The heaTC and the thermal hysteresis ΔThys can be obtained. The values of TC and ΔThys were determined from the maximum in the first derivative of the heating and cooling curves in an applied magnetic field of 0.01 TThe magnetic properties were measured in a Superconducting Quantum Interface Device (SQUID) magnetometer (Quantum Design MPMS XL) using the RSO mode in the temperature range of 5–370 K with a sweep rate of 2 K/min and vibrating sample magnetometer (VSM) (Quantum Design VersaLab) in the temperature range of 300–600 K with a sweep rate of 10 K/min. From the magnetic measurements, the ferromagnetic transition temperature xFex2−Py1−Siy quaternary compounds. Four possible lattice structures are found in the MnxFex2−Py1−Siy compounds. The orthorhombic Co2P structure (Pnma) forms in the Si poor region (y < 0.15). The single hexagonal Fe2P structure (P-62m) of y ≤ 0.5. For a Si composition of 0.5 ≤ y < 1.0, a three-phase region is observed, which consists of the Fe2P structure, the hexagonal Mn5Si3 structure (P63/mcm) and the cubic Fe3Si structure (Fm-3m) [A structural analysis has been performed to refine the lattice structure of the Mn (Fm-3m) .y ≤ 0.6 was chosen to synthesise samples MnxFex2−Py1−Siy with a hexagonal Fe2P lattice structure. The investigated range of silicon concentrations has been restricted to avoid the appearance of the orthorhombic lattice structure when the silicon concentration is too low. In contrast, if the silicon concentration is too high, the three-phase region is entered (with the Fe2P-type main phase and Fe3Si-type and Mn5Si3-type impurity phases), resulting in a decrease in the phase fraction of the main phase [A silicon concentration range of 0.33 ≤ in phase .a and c in the hexagonal main phase of the MnxFex2−Py1−Siy compounds is shown as a function of composition, refined values are given in x > 1 the lattice parameter a expands for increasing Mn and Si concentrations  In this case, the 3g site is fully occupied by Mn and the 3f site is fully occupied by Fe. The lattice parameter c is found to shrink for increasing Fe, Mn or Si concentration. The c/a ratio ranges from 0.54 to 0.58 and shows a similar dependence on composition as lattice parameter c  compounds was investigated by magnetisation measurements and is shown in TC increases with increasing Fe and Si contents. Materials with a low thermal hysteresis ΔThys can be found for both Fe-rich and Mn-rich compositions.The ferromagnetic-to-paramagnetic transition temperature xFex2−Py1−Siy compounds cover a broad range of ferromagnetic transition temperatures [TC was found to range from 65 to 470 K. The highest ferromagnetic transition temperature appears in the compound Mn0.3Fe1.7P0.6Si0.4 (TC = 470 K). This transition temperature is too high for magnetic refrigeration, magnetic heat pumping or waste heat conversion applications near room temperature. According to the trend of the phase diagram, it is clear that the transition temperature can be tuned higher if we increase the Si concentration. Therefore, part of the phase diagrams is marked ‘High TC’ as it shows a ferromagnetic transition temperature beyond the application range (TC > 470 K). The compounds Mn1.7Fe0.3P0.6Si0.4 shows the lowest ferromagnetic transition temperature (TC = 65 K) in the MnxFex2−Py1−Siy phase diagram. In general, the highest transition temperatures are found for low Mn and high Si contents, while the lowest are found for low Fe and Si contents.The Mneratures . In thisThys covers a broad range of values from 0 to 90 K. It is interesting to note that the largest hysteresis was found for a metal ratio of Fe:Mn ≈ 1:1 (x ≈ 1), corresponding to a full occupancy of the 3g site by Mn and the 3f site by Fe. The thermal hysteresis decreases with decreasing Fe, Mn or P content. The thermal hysteresis of the MnxFex2−Py1−Siy compounds is further tuneable by optimising the stoichiometry [The thermal hysteresis Δhiometry .xFex2−Py1−Siy compounds in the unstable region . Compared to ferromagnetic Fe2P-type samples  was chosen to synthesise MnxFex2−Py1−Siy samples with a hexagonal Fe2P-type lattice structure. The investigated range of silicon concentrations has been restricted to avoid the appearance of the orthorhombic lattice structure when the silicon concentration is too low. In contrast, if the silicon concentration is too high, a three-phase region is entered (consisting of the Fe2P-type main phase and the Fe3Si-type and Mn5Si3-type impurity phases), resulting in a decrease in the phase fraction of the main phase [2P-type main phase and the Fe3Si-type [5Si3-type impurity phases are shown in xFex2−Py1−Siy compounds (x < 1) is the Fe3Si-type phase. 3Si-type impurity increases continuously for increasing Fe and Si concentrations. For high Mn and low Si concentrations, the Mn5Si3-type impurity phase is dominant  to 1.21 (z = 0.09) results in an increase in transition temperature of 52 K and a decrease in thermal hysteresis of 10 K. These changes are caused by a variation in the amount of impurity phase and a shift in Mn, Fe ratio and P, Si ratio [0.7Fe1.3P0.6Si0.4 has about 6% Fe3Si impurity.  is associated with a large heat of transformation and a large hysteresis. For applications we like to have large magnetic entropy changes that can be boosted by contributions from heat of transformation, at the same time thermal hysteresis exceeding the adiabatic temperature change can’t be utilized in simple magnetisation and demagnetisation cycles. Therefore, these two parameters as important characteristics are compared to 1.3Fe0.7P0.5Si0.5 and Mn1.3Fe0.7P0.4Si0.6. These two samples both show a relatively large sharp peak, and the heat of magnetic transformation ΔmQ can be obtained by integrating the peak area. Note that this specific-heat peak contains all thermal effects involved in the magnetic phase transition. The ΔmQ of Mn1.3Fe0.7P0.5Si0.5 and Mn1.3Fe0.7P0.4Si0.6 compounds is 4.72 and 4.58 (J g−1), respectively.c/a ratio of the lattice parameters for the Fe2P-type hexagonal main phase of the MnxFex2−Py1−Siy system observed. As shown in c/a ratio starts from 0.5455 (Mn0.5Fe1.5P0.4Si0.6) and ends at 0.5826 (Mn0.9Fe1.1P0.67Si0.33). Most samples display no hysteresis when the c/a ratio is smaller than 0.57. For instance, sample Mn1.5Fe0.5P0.6Si0.4 is a sample without thermal hysteresis (c/a = 0.5696) and sample Mn0.5Fe1.5P0.6Si0.4 is a sample with a 2.6 K thermal hysteresis (c/a = 0.5701). In the grey region 0.56 < c/a < 0.57 we find both samples with and without hysteresis, and when the c/a ratio is larger than 0.57a steep increase in hysteresis with increasing c/a ratio. For instance, sample Mn0.7Fe1.3P0.5Si0.5 is a FOMT sample with about 12 K thermal hysteresis (c/a = 0.5701). The red line is a linear fit to these data.A clear correlation becomes visible when the thermal hysteresis is compared to the c/a ratio of the lattice parameters . For each temperature, the hysteresis increases with increasing Mn and Si concentration. Then, the hysteresis drops after it reaches a peak for a Mn content of x ≈ 1.0 and a Si content of y ≈ 0.45. Promising candidates can only be found in the grey area in x < 0.7, y < 0.4) and the Mn-rich/Si-rich part  of the phase diagram.As an example for the working temperature range of a thermomagnetic motor beeing between 20 °C (293 K) and 60 °C (333 K), with an optimal xFex2−Py1−Siy system has been established as a guide to find suitable materials for energy conversion applications in a thermomagnetic motor. We find a strong correlation between thermal hysteresis and c/a ratio, however, there are a wide range of samples with low (<2 K) or absent hysteresis that yet display large heat of magnetic transformation. Both Mn-rich with a high Si and Fe-rich samples with a low Si concentration were found to show a low hysteresis that can form promising candidates for applications in a thermomagnetic motor. It appears that Mn-rich samples are most suited for applications well below room temperature, while Mn-poor samples can be utilised at higher temperatures. The interesting experimental relationship between thermal hysteresis and the c/a ratio deserves further study. Furthermore, reducing the impurity phase fraction by optimising stoichiometry is important in lowering thermal hysteresis.A full-range magnetic phase diagram of the Mn


Congenital PAX6-aniridia is a rare panocular disease resulting from limbal stem cell deficiency. In PAX6-aniridia, the downregulation of the retinol-metabolizing enzymes ADH7 (All-trans-retinol dehydrogenase 7) and ALDH1A1/A3  have been described in limbal epithelial cells (LECs) and conjunctival epithelial cells. The aim of this study was to identify the role of retinol derivates in the differentiation of human LEC and its potential impact on aniridia-associated keratopathy development. Human LEC were isolated from healthy donor corneas and were cultured with retinol, retinoic acid, or pan-retinoic acid receptor antagonist (AGN 193109) acting on RARα, β, γ  or were cultured with pan-retinoid X receptor antagonist (UVI 3003) acting on RXR α, β, γ . Using qPCR, differentiation marker and retinoid-/fatty acid metabolism-related mRNA expression was analysed. DSG1 (Desmoglein 1), KRT3 (Keratin 3), and SPINK7  mRNA expression was downregulated when retinoid derivates were used. AGN 193109 treatment led to the upregulation of ADH7, KRT3, and DSG1 mRNA expression and to the downregulation of KRT12 (Keratin 12) and KRT19 (Keratin 19) mRNA expression. Retinol and all-trans retinoic acid affect some transcripts of corneal LEC in a similar way to what has been observed in the LEC of PAX6-aniridia patients with the altered expression of differentiation markers. An elevated concentration of retinol derivatives in LEC or an altered response to retinoids may contribute to this pattern. These initial findings help to explain ocular surface epithelia differentiation disorders in PAX6-aniridia and should be investigated in patient cells or in cell models in the future in more detail. Retinoids act through nuclear receptors at the transcriptional level; however, the non-receptor-mediated functions of retinoids have not been extensively studied as of yet . BesidesAll-trans retinoic acid (at-RA) acts through binding to the retinoic acid-receptors RAR α, β, γ , RXR α, β, γ , and PPARα, β/δ, γ .PAX6+/-) suffer from complete or partial absence of the iris tissue and corneal opacification and vascularization due to a limbal stem cell deficiency and several other ocular and systemic pathologies. An abnormal epidermal- (skin) like phenotype of the corneal epithelium is associated with severe ocular surface disease in congenital aniridia. These changes in the corneal epithelium are associated with PAX6 downregulation [Patients with a haploinsufficiency of the PAX6 (Paired box protein Pax-6) gene , Sox2 (transcription factor SOX-2), TFIID (transcription factor II D), pRb (retinoblastoma-associated protein), and MitF  . In Pax6ructures .ADH7 , ALDH1A1 , ALDH3A1 , PPARG (peroxisome proliferator-activated receptor gamma), FABP5 (fatty acid-binding protein 5), CYP1B1 (cytochrome P450 1B1), CYP26A1 (cytochrome P450 26A1), STRA6 (receptor for retinol uptake STRA6), and RDH10 (retinol dehydrogenase 10) [We have evidence that retinoic acid signaling may also be impaired in aniridia-associated keratopathy. The regulation of the transcriptional genes involved in the enzymatic conversion of retinoids as well as in retinoid- and fatty acid signaling is different in aniridia patient-derived ocular surface cells. These genes include nase 10) ,10.) TheBy manipulating genes at-RA levels, we sought to identify the genes that are related to the differentiation processes that are under the control of retinoic acid receptors or that are partly regulated by retinoic acid receptors.Since the effect of retinoids at the transcription level has not been studied on limbal epithelial cells so far, we provided the first profile of a set of transcripts to generate an idea as to what extent retinoic acid metabolism and signaling could influence AAK-related expression profiles or which transcripts are sensitive to at-RA treatment.Limitations: The experimental design of this study was not able to gain mechanistic insight into how PAX6 and retinoic acid directly interact with each other and how these effect the expression of their downstream genes. While working with primary cells with limited availability and low dividing capacity, it was also not possible to observe putative, short-term expression changes or PAX6 regulation in this setting.Ethical considerations: The use of patient tissue in this study was approved by the Ethical Committee of Saarland/Germany (no. 226/15).Limbal tissue of healthy corneal donors was punched out using a 1.5 mm biopsy punch. The tissue was incubated with 500μL collagenase A  for 24 h at 37 °C. Primary limbal epithelial cell (LEC) clusters were filtered and were dissolved with 2.5 mL trypsin-EDTA-solution . Then, LEC were resuspended in 500 μL keratinocyte serum free medium  and were seeded in one well of a 24-well plate. U LEC were incubated at 37 °C until confluence was reached. Thereafter, to transfer the LEC into a 6-well-plate, the cells were trypsinated and were dissolved in 3 mL of KSFM . The cells were passaged three times.After growing to confluence in four 6-wells, each individual LEC culture was treated for 24 h with 0.5 µM or 1.5 µM retinol (Ret) or all-trans-retinoic acid (at-RA), respectively. The concentration used for the 48 h treatment was 1 µM or 5 µM retinol (Ret) or all-trans-retinoic acid (at-RA) (Sigma-Aldrich GmbH). Moreover, the LECs were treated for 24 h or 48 h with 0.5 µM, 1.0 µM, or 1.5 µM of RAR antagonists (AGN 193109) or 0.5 µM, 1.0 µM, or 1.5 µM of RXR antagonists (UVI 3003) (Sigma-Aldrich GmbH). Ret, at-RA, AGN 193109, and UVI 3003 were dissolved in dimethylsulfoxid (DMSO) and were diluted in KSFM to reach the above concentrations. The control cultures were treated using DMSO during the same incubation periods. Each treatment and each incubation time  was repeated three times with individual limbal epithelial cultures from different donors. The incubation happened under the same conditions as the cell culture procedures described above.Cell viability was evaluated using the XTT assay as follows: human LECs were seeded into 96-well cell culture plates in KSFM. After reaching 70% confluence, the culture medium was changed to a culture medium containing either 0.1 µM, 0.5 µM, 1 µM, or 5 µM retinol (Ret), all trans-retinoic acid (at-RA), AGN 193109 (AGN), or UVI 3003 (UVI) for 24 h and 48 h, respectively. Then, freshly prepared XTT solution was added to each well. As a negative control, XTT solution was added to a well without cells. After an incubation period of 30–60 min, absorbance was measured at 550 nm using a 96-well microplate reader (TECAN Infinite F50).RNA and protein were extracted using the RNA/DNA/Protein Purification Plus Micro Kit  following the standardized protocol. The RNA-concentration was measured by means of UV/VIS-spectrophotometry . For protein concentration measurements, a standardized Bradford test (Sigma-Aldrich GmbH) was conducted. The cDNA-synthesis was performed using the OneTaq RT-PCR Kit . An amount of 500 ng of RNA was used for one reaction (50 µL of the cDNA-synthesis).ΔΔCt) was calculated. The following steps were repeated 40 times: initial denaturation to 95 °C for 5 min, denaturation at 95 °C for 10 s, and primer hybridization at 60° for 30 s. Elongation occurred when the sample was being heated up for the denaturation phases. The software QuantStudio™ Design & Analysis  was used for evaluation.For the qPCR, 1.25 µL Primer   or with 0.1 µM, 1 µM, 1.5 µM, or 5 µM retinol (Ret), all trans-retinoic acid (at-RA), AGN 193109 (AGN), or UVI 3003 (UVI) for 24 h and 48 h, respectively.During each step, the treatments were mixed in a total volume of 50 µL per well. After reaching confluence, the cells were directly fixed in 96-wells with 4% paraformaldehyde . Cells were permeabilized with ice cold pure methanol for 5 min. Nonspecific epitopes were blocked with Phospahte buffered saline (PBS) containing 5% fetal calf serum (FCS) and 0.3% Triton-X-100 for 2 h. Primary antibodies were diluted in PBS containing 1% FCS and 0.1% Triton-X-100. For staining, mouse anti-PAX6 antibody  was incubated overnight at 4 °C. Cells were washed three times with PBS containing 0.05% Tween. The secondary antibody, anti-mouse AF555 , was diluted at a ration of 1:200 in washing buffer. After 1.5 h of incubation at room temperature, the cells were washed three times again, and the nuclei were stained with DAPI . Cells were kept in PBS until image aquisition. Images were acquired with a Nikon Eclipse 100 microscope  equipped with a 20× objective and suitable filter sets . For each treatment, two images were processed with ImageJ software. To compare the control LECs and treated cells, exposure times and image processing  were performed identically. For semi-quantitative analysis, nuclei were identified using DAPI staining, and ROIs were defined with ImageJ Software; then, the mean integrated grey values for each cell were measured in the PAX6 (Cy3) channel. The total PAX6 intensity of the controls was set to 100%, and the signals were normalized to this value, and these were compared to those in the treated cells.For all of the treatment types , viability and mRNA expression values  were compared to the controls using one-way ANOVA followed by Dunnett’s multiple comparisons test. Analysis was performed with GraphPad Prism 7.04 software .In the primary limbal epithelial cell cultures in vitro, the highest proportion of the cells belonged to transient amplifying cells. In vivo, at the corneal surface, these cells would further differentiate into mature corneal epithelial cells. This differentiation process is limited in cell culture, but could be triggered by air-lift culture, etc. Nevertheless, changes in differentiation marker expression are already measurable in LEC cultures, and these can be useful in identifying factors influencing differentiation at the gene expression level. As a first step, we started to investigate the possible adverse effects of different treatment options on LECs.At-RA treatment for 24 h led to a minor but significant increase in viability at all concentrations, whereas 5 µM UVI led to a dramatic, significant decrease of cell viability. The same was true for the 48 h treatment using 5 µM AGN or UVI. There was no significant effect resulting from any of the other treatments on LEC viability, which was measured with the XTT assay .MKI76 mRNA expression is the highest in the M Phase of the cells [MKI67 was used as a cell proliferation marker. he cells ,12; therMKI67 expression at any of the incubation times  and 5 µM concentrations (FC = 0.001). AGN treatment for 24 h and 48 h led to significant MKI67 downregulation at the 1.5 µM concentration (FC < 0.2), and there was a less pronounced downregulation at lower concentrations. Nevertheless, 1.0 µM AGN treatment for 48 h showed no significant fold change reduction. UVI treatment for 24 h evoked significant MKI67downregulation when using the 1.0 µM (FC = 0.49) and 1.5 µM concentrations (FC = 0.16).Retinol (Ret) had no significant impact on on times . At-RA tPAX6 mRNA and protein expression measurements after at-RA and Ret treatment was necessary. The 24 h Ret treatment showed a significant but minor increase at the PAX6 mRNA level when using the 0.5 µM (FC = 1.2) and 1.5 µM Ret concentrations (FC = 1.2). There was a significant PAX6 mRNA downregulation when using the 1 µM (FC = 0.7) and 5 µM (FC = 0.6) concentrations in the at-RA treatment group after 48 h but not after 24 h, whereas neither AGN and UVI treatment nor any other conditions changed PAX6 mRNA expression  mice, which is an animal model for congenital aniridia. DSG1 expression was also downregulated in anirida patient-derived ocular surface cells when the siRNA-based PAX6 knockdown cell model was used [DSG1 (Desmoglein-1) is a cellular junction protein, and it has been reported to be downregulated in Seywas used ,13. SPINwas used . SPINK7 was used . It remaDSG1 mRNA expression tended to result in a reduction after 24 h Ret incubation, and DSG1 expression was significantly reduced after 48 h when using the 1 µM (FC = 0.06) or 5 µM (FC = 0.06) Ret concentrations. The treatments using the 1 µM or 5 µM at-RA concentrations also led to reductions in DSG1 (FC = 0.07 and FC = 0.3). In contrast, after treatment with the RAR antagonist AGN, DSG1 was upregulated after 24 h at all of the tested concentrations (0.5 µM (FC = 2.1); 1.0 µM (FC = 3.5); 1.5 µM (FC = 3.2)). The upregulation after the 48 h AGN incubation was not significant. DSG1 mRNA expression did not change significantly upon UVI treatment (SPINK7 mRNA expression did not decrease after the 24 h Ret treatment but decreased significantly after the 48 h Ret treatment with the 1 µM (FC = 0.3) or 5µM concentrations (FC = 0.3). The 24 h incubation with at-RA led to significant SPINK7 downregulation when using the 0.5 µM (FC = 0.52) and 1.5 µM concentrations (FC = 0.52) and to an even more prominent SPINK7 reduction after the 48 h at-RA treatment with the 1 µM (FC = 0.2) or 5 µM concentrations (FC = 0.1). AGN and UVI treatment had no significant impact on SPINK7 expression  and 1.5 µM concentrations (FC = 0.49) but not after the 24 h incubation time (KRT3 expression. KRT12 was significantly upregulated after the 24 h Ret treatment using the 1.5 µM concentration (FC = 1.4) and after 48 h with the 1 µM Ret treatment (FC = 1.7). A similar pattern was observed after the at-RA treatment, with a KRT12 upregulation occurring when the 1.5 µM concentration (FC = 1.7) was used after 24 h (KRT12 upregulation at the 1 µM concentration (FC = 2.8) was observed. AGN treatment for 24 h (FC = 0.5–0.7) and 48 h (FC = 0.3–0.7) led to a significant KRT12 downregulation when using all of the concentrations. UVI treatment did not lead to significant changes.ion time . The 24 ter 24 h . After tKRT19 mRNA expression was only significantly upregulated after the 48 h at-RA treatment when using the 5 µM concentration (FC = 2.9). KRT19 downregulation following AGN treatment was observed after 24 h when using the 0.5 µM (FC = 0.6) and 1.0 µM AGN concentrations (FC = 0.7).Enzymes and components of retinol and fatty acid metabolism  were altered on the ocular surface cells from aniridia patients and in the siRNA-based aniridia cell model. Our purpose was to monitor the gene expression changes of these genes in response to at-RA and pan-RAR and pan-RXR antagonists. This should allow us to draw conclusions regarding the observed expression patterns in the cells of aniridia subjects and in aniridia cellular models are affected by retinoid metabolism and which pathways are likely involved. Several transcripts were significantly altered upon treatment: See the results of transcripts ADH7, ALDH1A1, RBP1, RDH10, and CRABP2  and the MKI67 mRNA expression , there was a weaker or no response after 24 h compared to after the 48 h Ret treatment , which could partly be explained by the fact that Ret first needs to be converted to at-RA in the LECs. The presence of all of the enzymatic components converting retinol to active at-RA in corneal epithelial cells and the enzymatic conversion of retinol to at-RA has been previously observed in corneal tissue .PAX6 promotor does not contain functional retinoic acid response elements [−/−PAX6 rats, ALDH1A3 is not expressed, and it has been speculated that ALDH1A3 (RALDH-3) is a downstream target of PAX6 [PAX6 and retinoid signaling have already been linked previously, but these were mostly discussed and observed in the developmental processes of the eye and brain ,22. In n of PAX6 .+/- epithelial cells that had been derived from patients and in cellular models. For example ADH7, ALDH1A1, and ALDHA1A3 are downregulated in PAX6+/−-derived patient cells [In prior experiments, we observed an enrichment of deregulated genes that are associated with retinoid metabolism in PAX6nt cells ,10. Sincnt cells ,25 and snt cells , we specDSG1 are downregulated in aniridia mouse models and upon PAX6 knockdown in primary corneal epithelial cells [In addition to the changes in RA-associated transcripts, differentiation makers of the corneal epithelium such as al cells ,27.PAX6 mRNA expression, and 48 h of at-RA treatment was related to a downregulation of PAX6 mRNA. With the immunfluoresence imaging of nuclear PAX6 staining, we did not detect an effect of Ret treatment on the PAX6 protein level under any treatment condition  mice [PAX6 knockout [DSG1 downregulation happens due to a disturbed retinol metabolism and that DSG1 is not directly regulated by PAX6. The evidence for this would be as follows:/−) mice , in primknockout , in oculknockout .The dataDSG1 mRNA. Admission of the AGN antagonist (RAR receptor antagonist) led to a DSG1 mRNA expression increase  after 24 h with RAR antagonist treatment may be due to revertion of the unknown mechanism of mRNA stabilization.An upregulation of anscript . SignifiIn conclusion, a minor impact of at-RA treatment on keratin expression can be observed in LECs. The role of at-RA in the regulation of keratins and its role in AAK pathogenesis cannot be be drawn from in vitro LEC data in this study.In contrast to the relative clear changes in DSG1, SPINK7, or keratin expression patterns, ADH7, ALDH1A1, RBP1, RDH10, and CRABP2 expression  or ELOVLWhen applying higher concentrations of at-RA, there is also a risk for activating cis-RA-related signaling, as cis-RA is always present as an unavoidable contamination in at-RA solutions and could lead to over-activation of the RXR-RAR heterodimer . In addiExtensive titration of the concentations helps to avoid cross activity and supports the use of more specific agonists and antagonists. However, there is then a demand for an extensive use of primary or other cells. As these are not available, other tools beyond drug treatment should be prefered in the future to study the impact of at-RA signaling on gene expression in limbal epithelial cells.Expression of retinoic acid binding proteins CRABP2, RBP1, and retinol dehydrogenase RDH10 may change as a response to altered retinoid levels or may be influenced by activated RAR and RXR receptors.ADH7 mRNA expression, in contrast to the siRNA experiments reported by us, was not significantly regulated when at-RA was administered alone, but followed the same trend for some concentrations after 48 h with the 1 µM Ret or 1 µM at-RA treatment. Nevertheless, when using a higher Ret or at-RA dosage, the changes were even less pronounced . Therefore, we also checked the deregulated transcripts in the fatty acid metabolism.Changes in FABP5, PPARG, and ELOVL7 transcription, which are often observed in anirida, could be explained through the crosstalk of fatty acids with retinoic acid signaling. Responses of these latter-mentioned transcripts to at-RA could give valuable hints for such a crosstalk. FABP5 and PPARG may both effect at-RA and fatty acid (FA) signaling ,35, and ELOVL7, FABP5, and PPARG transcripts responded to at-RA and AGN.The functional balance between at-RA and fatty acid metabolism and signaling could be important to maintain an ocular surface epithelial phenotype without keratinization and cornified envelope formation. However, such a connection to at-RA signaling could not be established in our in vitro systems, which only anlaysed the short-term impact of at-RA concentration changes, although the DSG1 and SPINK7 mRNA expression, and some concentrations probablyt evoke reduced ADH7 expression. In the corneal and conjunctival epithelial cells of PAX6+/- patients, DSG1, ADH7, and probably SPINK7 downregulation could be evoked by elevated Ret or at-RA levels. A more global regulation of at-RA signaling that could interfere with changes in PAX6 expression is likely involved but is not adressed in the presented data.Summarizing all of the described data, we conclude that the influence of Ret or at-RA on gene expression in aniridia LECs exists independently from direct changes in PAX6 mRNA or protein expression. Our in vitro data suggest that an elevated Ret or at-RA level correlates with reduced At-RA signaling, which influences tissue-specific controled genes, is not understood in detail. The binding of RAR and RXR to specific promotor regions and associated chromatin remodeling as well as crosstalk to other signals such as FGF/MAPK, TFGβ/BMP, hedgehog, Notch Wnt, and Jak/STAT pathways are reported in other systems that are already separate from the eye . TherefoDSG1, SPINK7, and to lesser extent, the Keratins KRT12 and KRT3.Despite the limitations of our in vitro systems and the restrictions for transcriptional analysis, we were able to provide supporting data that retinoic acid signaling impacts several transcripts that are markers for corneal epithelial differentiation. These include


Testis-specific histone variants are crucial to promote open chromatin structure to enable nucleosome disassembly in the final stages of spermiogenesis. However, even after histone replacement, mature sperm retain a proportion of these variants, the function of which is unknown. The present study aimed to understand the functional relevance of the retained H2B and H2A variants, TH2B and TH2A. While no literature is available on the phenotype of TH2A knockouts, TH2B/TH2A double knockout male mice are reported to be infertile. In this study, ChIP-seq analysis was done for TH2B and TH2A to understand the epigenomics of the retained TH2B and TH2A, using murine caudal sperm. Distribution across genomic partitions revealed ∼35% of the TH2B peaks within ±5 kb of TSS whereas TH2A peaks distribution was sparse at TSS. Gene Ontology revealed embryo development as the most significant term associated with TH2B. Also, based on genomic regions, TH2B was observed to be associated with spindle assembly and various meiosis-specific genes, which is an important finding as TH2A/TH2B DKO mice have been reported to have defective cohesin release. A comparison of mouse and human TH2B-linked chromatin revealed 26% overlap between murine and human TH2B-associated genes. This overlap included genes crucial for embryogenesis. Most importantly, heterogeneity in the epigenetic landscape of TH2A and TH2B was seen, which is intriguing as TH2B and TH2A are well reported to be present in the same nucleosomes to promote open chromatin. Additionally, unlike TH2B, TH2A was enriched on the mitochondrial chromosome. TH2A was found to be associated with Nuclear insertion of Mitochondrial DNA sequences (NUMTs) in sperm. A comprehensive analysis of these observations indicates novel functions for the sperm-retained TH2B and TH2A. Mature sperm and round spermatids harbor the same haploid genome but different epigenomes. Developmental limitations of embryos resulting from somatic cell nuclear transfer (SCNT) and round spermatid injection (ROSI) as compared to sperm borne embryos reveal the importance of the unique sperm chromatin in efficient embryogenesis . The negNucleosome, being an octamer, contains two copies each of H2A, H2B, H3, and H4 draped by two turns of duplex DNA . The porGerm cell-specific variants of H2B and H2A, namely, TH2B (Hist1h2ba) and TH2A (Hist1h2aa), were first reported in mammalian testis . SubsequCrystal structure analysis comparing nucleosomal core particles (NCP) revealed that TH2A/TH2B containing nucleosomes have decreased hydrogen bonds and fewer DNA-histone contacts as compared to canonical counterparts. This leads to a more open chromatin in TH2A/TH2B nucleosome , 2017. IThe TH2A/TH2B duo is crucial for spermatogenesis and chromatin structure. Double knockout mouse of TH2A and TH2B show male sterility owing to loss of cohesin release during interkinesis and defects in transition protein 2 (TP2) incorporation and histone replacement . These lad libitum. For experiments, mice were euthanized via cervical dislocation.BALB/c-129-E mice (8–12 weeks old) were used in this study. Mouse husbandry and experimental procedures were carried out as per the guidelines of the National Institute for Research in Reproductive Health Institutional Animal Ethics Committee (NIRRH-IAEC). All animal experiments were approved by the NIRRH-IAEC (project no. 07/18). Mice were housed in groups of four per cage under conditions of 12-h light–dark cycle and provided with water and food g*30 min at 4°C to eliminate somatic cells, if any. Quantity and purity of sperm was determined using Neubauer chamber. On an average, sperm yield per mouse  was ∼5–6 million.Mouse sperm were isolated as per the protocol described by g for 10 min at 4°C. The pellet was washed thrice by centrifugation as above and resuspended in 250 µl of 2D lysis buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 0.1 M DTT and protease inhibitor cocktail (Roche).Western blot analysis of TH2A and TH2B was done to detect their presence in mouse caudal sperm (mature sperm). Whole testicular cell lysates were used as positive control. Towards this, after removing the tunica albuginea, the seminiferous tubules were teased out in PBS and incubated at 37°C for 1 h at 70 rpm. Following this, the testicular cell suspension was filtered through 40-µm Falcon™ Cell Strainers (Fisher scientific) and pelleted by centrifugation at 2,000g for 10 min at 4°C, pellet washed as described above, and resuspended in 150 µl of 2D lysis buffer. Caudal sperm suspension and whole testicular cell lysate were incubated on a rotary shaker overnight at 4°C. The following day, the lysates were subjected to bead beating homogenization in a Fast prep-24 homogenizer . The homogenates were incubated on ice for an hour and then centrifuged at 16,000 g for 30 min at 4°C. The supernatants containing the protein lysate were quantified for protein content using the 2-D Quant Kit .Caudal sperm (∼8 million cells) collected by the sperm swim-up procedure were initially treated with 10 mM dithiothreitol (DTT) in PBS at room temperature for 1 h to release the protamines. The DTT pre-treated sperm were then pelleted by centrifugation at 2,000 trans-blotted on nitrocellulose membrane at 100 V for 1 h Non-specific binding to the membrane was blocked with 3% NFDM in 0.1 M PBS (blocking buffer) for 1 h at room temperature. Next, these membranes were incubated with primary antibody overnight at 4°C, followed by two washes with PBS containing 0.05% Tween 20 (PBST). The membranes were subsequently incubated with HRP-conjugated secondary antibody for 1 h at RT. The unbound antibodies were eliminated by washing the membrane thrice with 0.05% PBST. Chemiluminescence detection of protein of interest was done using Western Blot Chemiluminescence HRP substrate (Takara-bio), in ChemiDoc imager (Bio-Rad).For Western analysis, 200 µg of caudal sperm for TH2A and 100 µg for TH2B and 50 µg testicular cell lysates were resuspended in Laemmli buffer containing 25% 2-mercaptoethanol, 0.01% bromophenol blue, 50% Glycerol, 20% SDS, and 0.25 M Tris-HCl, pH 6.8. Samples were heated for 5 min at 95°C and subjected to 15% SDS-PAGE at 100 V for 2 h and Primary antibodies used were rabbit anti-mouse TH2A  at a dilution of 1:250 and rabbit anti mouse TH2B Antibody (Sigma-Aldrich #07–680) at a dilution of 1:5,000. Secondary antibody used was HRP-conjugated swine anti-rabbit IgG (Dako) at a dilution of 1:1,500 in 1% blocking buffer.The protocol described by g for 10 min at 4°C to pellet down the denser protamine-bound DNA, and the supernatant containing mononucleosomal chromatin was retrieved. Preclearing of the MNase-digested chromatin was done with blocked protein-A Sepharose beads  for 1 h. The chromatin (from 12 million sperm in two replicates) was then immunoprecipitated overnight with 6 µg each of either TH2A or TH2B antibody. The following day, blocked beads were added and incubated with end-to-end mixing at 4°C for 4 h, to capture the antibody chromatin complexes.The MNase digestion was stopped by addition of 0.5 M EDTA. The digestion mixture was centrifuged at 16,000 g for 2 min at RT. The eluted (∼300 µl) immunoprecipitated chromatin DNA was treated with 6 µl of RNaseA (10 mg/ml) at 37°C for 30 min and overnight incubation at 56°C with 6 µl of proteinase K (10 mg/ml) and extracted via column-based DNA extraction. Mononucleosomal DNA (one-tenth of the IP reaction) was saved as input. DNA quality and concentration were evaluated using Qubit Fluorometric Quantification (Invitrogen Qubit™ four Fluorometer #Q33226). The entire eluate was mixed with 6× DNA loading dye  and electrophoresed on a 5% polyacrylamide gel for 45 min at 100 V with 0.1 M TBE as electrode buffer. The gel was post-stained with ethidium bromide. Mononucleosomal DNA band at ∼150 bp was excised and the mononucleosomal DNA was re-precipitated and subjected to sequencing after pooling the replicates.The antibody–bead complexes were washed by centrifugation  and the chromatin was eluted in two rounds in Tris-EDTA buffer, pH 8.0, containing 10 mM Tris-HCl, 1 mM disodium EDTA, and sodium dodecyl sulfate (1%) by centrifugation at 11,000 www.bioinformatics.babraham.ac.uk/projects/fastqc): 1) Base quality score distribution, 2) Sequence quality score distribution, 3) Average base content per read, 4) GC distribution in the reads, 5) PCR amplification issue, and 6) Checking for overrepresented sequences (Ver-0.36) (Mus Musculus (mm10) release downloaded from Sanger Institute database. Alignment was performed using BWA MEM (Ver-0.7.12)  using HOMER (annotatepeaks)  indicates a range of 100–1,000 with majority of them being approximately 100–400. We have used BedSect V3 tool for peaks classification into common and unique peaks across each sample  (Agilent). Sequencing was performed with Illumina Hiseq using 150 bp paired end sequencing. For Input and TH2B ChIP-Seq libraries and for TH2A, ∼30 million and 34 million unique paired end reads were attained, respectively. The following parameters from fastq file were determined using FastQC (equences . Based oer-0.36)  to retai-0.7.12) . The ali-0.7.12) . Statisttepeaks) . The lenh sample .https://bioviz.org/)  was the same for both TH2A and TH2B graph tracks for comparisons. Heatmaps of different BigWig coverage ChIP signals for sperm PTMs (from GSE79227) on TH2B peaks were generated using Deeptools 3.3.2, computeMatrix (computeMatrix 3.3.2), and plotHeatmap (plotHeatmap 3.3.2) function in Galaxy server   was done for the enrichment of annotated peaks over the genomic features and the distance from TSS, and visualized using the GenomicDistribution package in the LOLAweb tool, which is based on LOLA R package (LOLAweb version: 2ddd6d682) . Assessmiz.org/) . Relativ3.2.0.1) . Mouse s3.2.0.1) . All the3.2.0.1) .http://chip-atlas.org), peaks associated with primordial germ cells (PGCs), spermatogonial stem cells (SSC), round spermatids, and sperm with a threshold significance of more than 200 were viewed on IGV Ver 2.8.13  in 25 μl of total volume. Two individual 10-μl reactions were set up in CFX96 Touch Real-Time PCR Detection System (Bio-Rad). The PCR conditions were as follows: 95°C for 3 min; 95°C for 30 s, appropriate annealing/extension and plate read for 30 s for 40 cycles, and melt curve analysis 75–95°C at 0.5°C increment at 5 s/step.The enrichment of gene regions identified to be associated with TH2A/TH2B by ChIP assays were confirmed by quantitative real-time PCR in triplicate. MACS2 peak specific primers were generated for TH2A-associated genes—and Mxi1 . Input wChIP DNA enrichment was calculated as the percent of input  using the formula:100*2^ (Adjusted Input—Ct (IP))http://bejerano.stanford.edu/great/public/html/index.php) version 4.0.4 was used for functional analysis of TH2A/TH2B-associated genomic regions  for Mouse: GRCm38  (Genomic Regions Enrichment of Annotations Tool (GREAT) (c. 2011) . The defhttps://www.ncbi.nlm.nih.gov/research/HistoneDB2.0/):Protein FASTA sequences for the following canonical and testis-specific histone variants were retrieved from HistoneDB2.0 . Coloring of the residues according to the hydrophobicity was also performed using Jalview .Histone variants differ from their replication-dependent canonical counterparts in terms of protein sequences and domains. This difference in sequences may ultimately lead to recruitment of entirely new post-translational modifications (PTMs) and novel interacting chromatin modeling enzymes or modifiers and consequently an altogether new epigenetic profile as compared to that of the somatic histone. Thus, it becomes crucial to closely study the amino acid sequences of these variants. Also, since mice is an experimental model for mammalian studies, to understand the evolutionary conservation of the variants TH2B and TH2A, their homology in protein sequences was assessed by pairwise global alignment of TH2B and TH2A in human and mouse. This revealed ∼95% identity between human and mouse TH2B and ∼89% homology between amino acid sequence of human and mouse TH2A, which differ mostly at the C-terminal . Within Hist1h2aa and Hist1h2ba genes were epigenetically profiled across the murine male germ cell stages, starting from PGCs to sperm using multiple ChIP-seq data from ChIP atlas  . We therIP atlas . Only hiIP atlas . ChIP-seIP atlas . In the H2A/TH2B .H3f3b and H1fnt. A profile similar to that of TH2A/TH2B was found for H3f3b (H3.3) whereas H1fnt, which is involved in spermiogenesis, predominantly showed H4ac and H3K9ac marks in round spermatids and H3K4me3 in sperm . A flow chart depicting a brief summary of the bioinformatics analysis is shown in www.bioinformatics.babraham.ac.uk/projects/goliath/). A chromosome wise coverage map was generated  of binary alignment files in Integrated Genome Browser (IGB)  revealedenerated . Consideenerated , an occuTH2B association was noted on all autosomes and sex chromosomes except the mitochondrial (MT) chromosome. However, unlike TH2B, a staggered association of TH2A binding loci with genic regions was seen, with binding sites only on chromosomes 1, 4–8, 10–13, 17–19, MT, X, and Y. A fair enrichment was seen on MT chromosome. Locus enrichment overlap analysis was done using LOLAweb . TH2A- acis-regulatory interactions of TH2A/TH2B with their associated loci) and GOliath (gene list-based Gene Ontology) were used for Gene Ontology analyses of the genomic regions for TBAG and TAAG lists  is an endodeoxyribonuclease, important for the resolution of Holliday Junctions (HJs), which is essential for segregation of chromosomes. Yeast Slx4’s mammalian ortholog, BTBD12 mutants are sub-fertile with almost under 15% normal spermatozoa  is an important meiotic synaptonemal complex protein part of axial elements/lateral elements. Male mutants of Sycp2 have defects in meiosis with failure in axial element formation and are sterile (Hus1b) cell cycle checkpoint gene required for genomic integrity , RIKEN cDNA 1110017D15 gene (1110017D15Rik), and testis, prostate, and placenta expressed (Tepp)  . This suTgif1, Mxi1, Zfp830, Thbd, Ppard, Dlk1, and Asf1b are also involved in various aspects of embryogenesis like blastocyst growth, development and hatching, embryo implantation, and gastrulation. From their pioneer study using sperm from fertile human donors, Hammoud et al. have shown that the retention of histones is not random but on loci of developmental importance, imprinted genes, and regulatory RNAs  and IMPC data. This search yielded six TBAGs—Epas1, Epc2, Lmbrd1, Msx1, Pitx2, and Slc40a1  were referred . Four em Slc40a1 . Moreove Slc40a1 . One hun Slc40a1 . Four THe testis . These me testis .Hoxa9, Rec8, Dhh, Nectin2, Sox30, Adam1a, and Msxi1—and TAAGs—2610005L07Rik, Tcte2, Mt-tp, and Mt-co1—were cross-validated by ChIP-qPCR (A set of TBAGs—hIP-qPCR . These fhttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE79227) were used for identifying activating histone marks (H3K4me3/me2 and H3K9Ac), super enhancer modifications (H3K27Ac and H3K4me1), and repressive marks . The genome-wide BigWig coverage tracks for mm10 were retrieved using ChIP atlas and these bigWig scores were submitted to Galaxy along with the TH2B genomic interval file. Using the computeMatrix function, a matrix file was generated for signals ±2 kb around the TH2B MACS2 peak. The computed matrix was then visualized using plotHeatmap. While repressive marks H3K27me3, H3K9me3, and H3K36me3 showed no overlap with TH2B chromatin, a positive enrichment of H3K4me2 and H3K4me3 overlapping the peak sequences was observed  with expression restricted to testis, nine genes  with biased testicular tissue expression, and eight ubiquitously expressing genes  with highest expression in testis. Only three genes  were identified with broad expression across all the tissues and highest expression in testis. Intriguingly, two enhancers  controlled the expression of six genes: 4921511H03Rik, Gm9758, Gm10354, Speer4d, Gm17019, and Speer4e, which have either biased or restricted testicular expression.A mild correlation was observed between active enhancer mark H3K27Ac and TH2B marked loci. Recent chromatin analysis studies on sperm have shown that H3K4me2 and H3K4me3 marks are both enriched in sperm, with many overlapping regions marked by these PTMs . Thus, ovia sperm H3K4me3  studies , 2017. PWhile the importance of TH2A/TH2B in histone eviction is well acknowledged, the significance of their retention in mature sperm is still unclear. The present study attempted to demystify the relevance of sperm-retained TH2A/TH2B.in silico via ChIP-seq data across germ cell stages . We obsvia separases and the sister chromatin cohesion is disrupted, followed by their pulling apart to the opposite poles via spindles. In the double mutant TH2A/TH2B mice, there is an incomplete release of cohesin at interkinesis and accumulation of secondary spermatocytes  accompanying TH2B marked loci is crucial to unravel the molecular events and significance of TH2B-associated chromatin. With respect to repressive marks H3K27me3, H3K9me3, and H3K36me3, there was almost no overlap with TH2B chromatin. We observed a positive enrichment of H3K4me3 overlapping with the peak sequences. Both H3K4me3and H3K4me2 were enriched . We alsoNr4a3, Osr1, and Zpr1 are important for gastrulation and blastocyst differentiation. N4bp2l2 is crucial for embryo development (It is important to extrapolate the findings in mouse to humans as the former is an experimental model. Also, in the literature as previously stated, there is a range in the histone retention percentage in sperm. For humans, it is 4%–10%, whereas for mice, it varies from ∼1% to 10%. These differences make it important to compare the chromatin occupancy of TH2B between the orthologs. Any conservation may be important from the point of evolution. To highlight the relevance of our findings, we explored the commonality in the dynamics of TH2B between murine and human sperm. A subset of genes important for morphogenesis and embryo development were found common between the two . Genes selopment . It is eSendler et al. have found sperm RNAs to be associated with H3K4me3 but not H3K27me3 . We see in silico analysis have attributed a myriad of novel functions to sperm-retained TH2B with respect to embryo development and spermatogenesis. Our data indicate a degree of conservation between the TH2B–DNA linkage across human and mouse. Our observations also convincingly demonstrate that TH2B/TH2A are relatively independent in their genome-wide chromatin associations. The TH2A–MtDNA link observed by us needs to be explored further. In vivo, validation of these observations will be crucial to further understand TH2B dynamics.In summary, our murine TH2A and TH2B ChIP-seq data and


Sphingomonas sp. PAMC 26621 (SpTrx). The aromatic cluster of SpTrx was comprised of W11, F26, F69, and F80, in which F26 at the β2 terminus was buried inside. The substitution of tyrosine for F26 changed the SpTrx conformation substantially compared to that of F69 and F80. Further biochemical and spectroscopic investigations on F26 showed that the F26Y, F26W, and F26A mutants resulted in structural instability of SpTrx in both urea- and temperature-induced unfolding and lower insulin reduction activities. The Trx reductase (SpTR) showed lower catalytic efficiencies against F26 mutants compared to the wild-type SpTrx. These results suggest that buried F26 is essential for maintaining the active-site conformation of SpTrx as an oxidoreductase and its structural stability for interactions with SpTR at colder temperatures.Thioredoxin (Trx), a small redox protein, exhibits thermal stability at high temperatures regardless of its origin, including psychrophiles. Trxs have a common structure consisting of the central β-sheet flanked by an aliphatic cluster on one side and an aromatic cluster on the other side. Although the roles of aromatic amino acids in the folding and stability of proteins have been studied extensively, the contributions of aromatic residues to the stability and function of Trx, particularly Trxs from cold-adapted organisms, have not been fully elucidated. This study examined the roles of aromatic amino acids in the aromatic cluster of a Trx from the psychrophilic Arctic bacterium  Thus, the modified Stern-Volmer equation (F0/F = 1+ KD[Q]) was used in these cases, where Q is the acrylamide concentration, and KD is the Stern-Volmer constant calculated from the slope of the linear region of the quenching curve.For the acrylamide-induced quenching of protein fluorescence measurements, proteins were incubated at various temperatures  for 2 h in buffer B. After incubation, acrylamide (0–0.5 M) was added to buffer B for 2 min at 25°C, and the fluorescence was measured. Acrylamide Stern-Volmer plots are presented as the ratio of intrinsic fluorescence intensity in the absence of acrylamide  for 2 h before being added to the reaction mixture containing 10 μM bis-ANS in buffer C. After 30 min of incubation, the fluorescence spectra of the samples were measured at 25°C using a Scinco FS-2 fluorescence spectrometer (excitation at 385 nm and emission at 400–600 nm).Tm value, at which 50% of the protein was unfolded, was determined using Protein Thermal Shift software v1.4 (Applied Biosystems).A thermal shift assay using SYPRO orange dye was performed on an Applied Biosystems StepOnePlus real-time PCR instrument in the 25–99°C range in 1°C/min increments. The proteins (0.6 mg/mL) were added to a phosphate buffer  containing 10× SYPRO orange dye in a total volume of 20 μL. The The samples at a final concentration of 0.3 mg/mL in buffer B were incubated at different temperatures  for 2 h. The CD spectra were measured using a JASCO J-1500 spectropolarimeter at the Korea Basic Science Institute . The results show residual ellipticity (mdeg) versus wavelength (nm) using GraphPad Prism software. The αα-helical content of each protein was calculated using the K2D3 server .Km) and the catalytic rate constant (kcat) were calculated by using the double reciprocal Lineweaver–Burk plot.The assay mixture , and 180 mM NAPDH) was incubated with 50 nM SpTR and a range of Trxs (0.1–10 μM) at 4 or 30°C for 3 min. The SpTR activity was measured from the reduction of DTNB at 412 nm. One enzyme unit is defined as the amount of enzyme catalyzing the formation of 1.0 μM of 1,3,5-trinitrobenzene per min. The Michaelis–Menten constant (Insulin (0.13 mM) and an appropriate amount of SpTrx proteins were added to the reaction mixture  to a final volume of 800 μL. A negative control was set up without the Trx protein. The mixtures were incubated at 25°C and measured at 10 min intervals for up to 100 min by recording the absorbance of the precipitated β-chain of insulin at 650 nm using a Shimadzu UV-1800 spectrophotometer .Acetobacter aceti Trx (54.2% amino acid sequence identity) showed a typical Trx fold with five β-strands sandwiched on both sides by a pair of α-helices  as soluble proteins. The proteins were purified by nickel-chelate affinity chromatography, followed by Capto Q anion-exchange chromatography. SpTR was purified further to homogeneity by Superdex 200 gel filtration chromatography (The recombinant SpTrx (107 amino acids) and SpTR (321 amino acids) proteins with a C-terminal six histidine-tag are expressed in tography . SpTrx atography .The temperature-induced unfolding of SpTrx and SpTR was evaluated by measuring the intrinsic protein fluorescence (excitation at 280 nm) upon incubation of proteins at 4–70°C for 2 h, respectively. SpTrx has four tryptophan and no tyrosine residues, whereas SpTR has four tryptophan and eight tyrosine residues. Although SpTrx maintained its tertiary structure within the 4–70°C range, SpTR began to lose its tertiary structure from 50°C onwards and lost its structure at 60°C . The opt0/F value , suggesting that the presence of a tyrosine hydroxyl group at positions 26 and 80 caused structural instability  on the structural stability of SpTrx were investigated further by generating two more F26 mutants (F26W and F26A) by site-directed mutagenesis . AlthougTm values for the F26 mutants than WT (73.4°C): 63.5°C for F26Y, 59.3°C for F26W, and 56.4°C for F26A  for 2 h. WT, F26Y, and F26W maintained their overall tertiary structures in the 4–50°C temperature range . On the for F26A . The Tm 0/F value at 50°C than WT and F26W. The inverse Stern-Volmer quenching constant (KD-1) of F26Y decreased from 0.43 M at 30°C to 0.36 M at 50°C  for 2 h before measuring the CD spectra. WT, F26Y, and F26W maintained their secondary structures in the 4–90°C temperature range with similar α-helical content (approximately 68–71%), whereas F26A exhibited increased α-helical content (80.5%) at 4°C and underwent a substantial structural change at 90°C with a 75.0% α-helical content .Km values to SpTrx WT, F26Y, and F26W . On the other hand, the kcat values of SpTR for F26Y and F26W were 46% and 31% lower relative to the kcat value for WT (137.2 min-1), respectively, which led to lower kinetic efficiencies of SpTR for F26Y and F26W. SpTR showed a 10-fold higher Km value to F26A compared to the other mutants. At 4°C, SpTR showed approximately 50% maximal activity  or mesophilic Cutibacterium acnes (CaTrx) [Multiple sequence alignment  showed t (CaTrx) , 31. AltSubtle adjustment of the aromatic residues in the enzyme active sites is crucial to maintaining proper conformations of cold-adapted enzymes at colder temperatures despite their intrinsically flexible structures –35. For Tm value of 73.4°C for SpTrx compared to approximately 123°C for thermophilic Trxs [E. coli [The present study showed that SpTrx and SpTR co-evolved with distinct cold adaptation patterns during evolution, and this pattern of heat-stable Trx and heat-labile TR has been observed , 14. Trxlic Trxs . The hig[E. coli . In cont[E. coli .In conclusion, this study sheds light on the roles of buried phenylalanine in the structural stability of SpTrx during adaptations to colder temperatures. Further studies on the co-evolution of TR, Trx, and downstream oxidoreductases will elucidate the cold adaptation mechanism of Trx systems in psychrophilic microorganisms.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 FigFluorescence intensities of oxidized SpTrx WT (A) and reduced SpTrx WT (B).(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


Tripterygium wilfordii, is a promising drug due to its anticancer and antiobesity activities. Although a previous study achieved friedelin production using engineered Saccharomyces cerevisiae, strains capable of producing high-level friedelin have not been stably engineered. In this study, a combined strategy was employed with integration of endogenous pathway genes into the genome and knockout of inhibiting genes by CRISPR/Cas9 technology, which successfully engineered multiple strains. After introducing an efficient TwOSC1T502E, all strains with genetic integration  showed a 3.0∼6.8-fold increase in friedelin production compared with strain BY4741. Through further double knockout of inhibiting genes, only strains GD1 and GD3 produced higher yields. Moreover, strains GQ1 and GQ3 with quadruple mutants  displayed similar increases. Finally, the dominant strain GQ1 with TwOSC1T502E was cultured in an optimized medium in shake flasks, and the final yield of friedelin reached 63.91 ± 2.45 mg/L, which was approximately 65-fold higher than that of the wild-type strain BY4741 and 229% higher than that in ordinary SD-His-Ura medium. It was the highest titer for friedelin production to date. Our work provides a good example for triterpenoid production in microbial cell factories and lays a solid foundation for the mining, pathway analysis, and efficient production of valuable triterpenoids with friedelin as the skeleton.Friedelin, the most rearranged pentacyclic triterpene, also exhibits remarkable pharmacological and anti-insect activities. In particular, celastrol with friedelin as the skeleton, which is derived from the medicinal plant  Quercus suber   . In S. cterpenes , such asterpenes , α-amyriα-amyrin , and oleα-amyrin .Friedelin is the most highly rearranged pentacyclic triterpene in plants, , the cyctHMG1), farnesyl diphosphate synthase (ERG20), squalene synthase (ERG9), and 2,3-oxidosqualene synthase (ERG1), which are involved in the mevalonate (MVA) pathway, as well as an uptake control transcriptional regulator (UPC2.1) and POS5 gene (encoded a NADH kinase), which participate in the auxiliary pathway. Overexpression of these genes represented an effective strategy for increasing the metabolic flow of terpenes according to the literature (BTS1 (geranylgeranyl diphosphate synthase), YPL062w, YJL064w, and ROX1 (repressor of hypoxia) on the competing pathways were knocked out by the CRISPR/Cas9 system , which was cultivated in YPD , 2% peptone (OXOID), and 2% glucose) medium. Recombinant yeast strains were selected on synthetic dropout (SD) medium  at 30°C. E. coli Trans1-T1  was used for plasmid construction, maintenance and amplification, and the bacteria were grown at 37°C in Luria-Bertani (LB) medium  with 100 μg/ml ampicillin or 50 μg/ml kanamycin if necessary.The initial strain used in this study was BY4741  technique was performed , and theThmg1, ERG20, ERG9, ERG1, and POS5) were downloaded from the Saccharomyces Genome Database (SGD) (yeastgenome.org). The gene sequence of UPC2.1 was based on Hu. et al. (UPC2.1-Blunt zero plasmid. DNA sequence information of genes is listed in S. cerevisiae strain BY4741 was extracted as a template to amplify the open reading frame (ORF) of overexpressed genes according to the instructions of the yeast genome extraction kit  Co., LTD.). According to the sequence information of genes , primers with a Type IIS restriction endonuclease BsaI recognition site (GGTCTCNNNNN) were designed. The promoter Ptdh3-Padh1, terminator TPI1t-PGIt, and genes Thmg1 and ERG20 were cloned by PCR with Phusion® High-fidelity DNA Polymerase  using plasmids P1, T1, and BY4741 as templates. PCR products were recovered by a Gene JET Gel Extraction Kit . The Golden Gate reaction was performed on the purified DNA fragments with T4 DNA ligase under the following conditions: 25 cycles of 37°C for 3 min and 16°C for 4 min, followed by 50°C for 5 min and 80°C for 5 min, and then held at 4°C. The reaction products were transformed into E. coli Trans-T1 competent cells and cultured at 37°C, and then assembled plasmids were sequenced by Tianyibiotech . The correct plasmid was stored, and overexpression module I was constructed. The results are shown in The first step is to clone the promoter sequence, gene, and terminator sequence. Overexpressed gene sequences -F/R; L1: L1 (15 site-1)-F/R; L4: L4-15 site2--F/R; 15 site2: 15 site2-F/R). The 15 site1, His, L1, L4, and 15 site2 fragments were cloned by PCR with Phusion® High-fidelity DNA Polymerase  using plasmids T1 and T3 and genomic DNA as templates. After the PCR products were recovered, overlap PCR was performed to construct head and tail homologous arms, and the results are shown in The second step is to construct homologous head and tail arms for integration. Primers were designed based on the sequence information for L1, L4, pESC-His and the S. cerevisiae BY4741 cells via electroporation. Competent cells were prepared according to the following protocol: single colonies were inoculated in 4 ml liquid YPD to OD600 = 0.6–1.0 and collected via centrifugation at 8,000 × g for 1 min. The obtained cell pellet was washed twice using 1 ml precooled sterile water and then incubated in 4 ml transformation reagent  for 20 min at 25°C. Conditioned cells were collected by centrifugation, washed twice using 1 ml ice-cold 1 M sorbitol buffer, and then resuspended to a final volume of 100 μL in sorbitol buffer. Cells with 500 ng DNA fragments were electroporated at 3 kV, 25 μF, and 200 Ω , incubated in 1 ml sorbitol buffer for 2 h at 30°C, and then plated on selective media for 2–3 days.Finally, these overexpression modules were integrated into the yeast genome in different combinations. For genomic integration, transcription unit modules, selective markers, and integrated homologous arm modules were transformed into BTS1, ROX1, YPL064w, and YJL062w were designed using an open-source tool http://yeastriction.tnw.tudelft.nl, and efficient target sequences were selected , the Cas9 expression vector p414-TEF1p-Cas9-CYC1t , and the general vectors pTY-U01 and pTY-U02 used for gene knockout were preserved in the laboratory. Specific gRNAs targeting selected . All gRNassembly . Similar62w-gRNA   and then spliced by overlapping PCR to obtain the homologous repair fragments of four genes. The primers can be found in URA3 labelling were removed with 5-fluoroorotic acid -mediated homologous recombination repair is required. Homologous repair fragments of tic acid , and Castic acid . The recMiFRS from Maytenus ilicifolia (PdFRS from Populus davidiana (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). And the codon-optimized plasmid pYES2-TwOSC1T502E from T. wilfordii was preserved in the laboratory (GenBank accession number: KY885467.1). These three constructed vectors were transformed into recombinant strains using the Frozen-EZ Yeast Transformation II Kit , and the pYES2 vector and pESC-Leu vector were transformed into yeast as a control. The transformants were screened on the corresponding solid plates and confirmed by colony PCR and DNA sequencing. Plasmid and sequence information is listed in icifolia  and PdFRavidiana   containing 30 ml of SC-His-Ura and SC-His-Leu medium containing 2% glucose and incubated with shaking at 200 rpm and 30°C for 2 d. Then, the cells were collected and induced in 30 ml of SC-Ura-His medium with 2% galactose in place of glucose and further cultured at 200 rpm and 30°C for 72 h. The dominant strain harbouring pYES2-Next, the extracts were dissolved in 1 ml hexane for analysis by gas chromatography and mass spectrometry (GC–MS). For analysis, a 1 μL sample was injected into an Agilent 7000 gas chromatograph in a nonshunt manner using a DB-5 ms capillary column  with a DB-5 ms  capillary column. One microlitre of the concentrated organic phase was then injected at a He flow rate of 1 ml/min with a temperature program of 1 min at 50°C, followed by a gradient from 50 to 260°C at 50°C/min and then to 305°C at 20°C/min, with a 15 min hold. The ion trap temperature was 230°C. The electron energy was 70 eV. Spectra were recorded in the range of 10–550 m/z. Standard chemicals were purchased from YuanYe Biotech .MiFRS gene in expression plasmid was introduced into the wild-type strain BY4741, the product friedelin reached only 0.39 mg/L  and isopentenyl diphosphate (IPP) to -geranylgeranyl diphosphate (GGPP), which would decrease the 2,3-oxidosqualene supply for triterpene production due to its high consumption of FPP and IPP -geranylgeraniol (GGOH) production, when UPC2.1 was integrated into the YPL062w deletion site in the strain BY-HZ13. Ro et al. found only a modest effect on amorphadiene production when overexpressed in the EPY208 background, which was not significantly improved . This phenomenon suggested that ERG20 was superior to POS5 for friedelin yield. As Gao reported in 2018, overexpression of POS5 gene in strain GW6 with ERG20/NADH-HMGr/PPDS-ATR1 expression cassette could enhance NADPH supply and produced protopanaxadiol 3∼4.55 fold higher under different sugar fermentations  strains GD1∼GD4 showed different trends of friedelin production  showed a similar effect, leading to a great increase in friedelin production in the as-constructed strains GQ1 and GQ3, a decrease in GQ2 and even death to GQ4. Notably, among the tested strains, the quadruple mutant GQ1 harbouring TwOSC1T502E had the highest friedelin production level (19.75 ± 2.78 mg/L) in the shake flask experiments, which was approximately 20-fold higher than that of the wild-type strain BY4741  was approximately 20-fold higher than that in the original strain. Next, the screened high-efficiency FRS TwOSC1T502E was introduced into the dominant strain GQ1 and cultured in optimized medium in shake flasks. The final production of friedelin was 63.91 ± 2.45 mg/L, which was approximately 65-fold higher than that of the wild-type strain and 229% higher than that in ordinary SD-His-Ura medium. To the best of our knowledge, the 63.91 ± 2.45 mg/L yield of friedelin based on shake flask production was the highest titer in heterologous production reported thus far. The engineered yeast strains constructed in this work not only provide new ideas for enhancing friedelin production but can also be used as chassis strains for the mining, pathway analysis, and efficient production of valuable triterpenoids with friedelin as the skeleton.In this study, a high-yielding friedelin engineered strain was constructed by comprehensive engineering strategies. First, multiple endogenous genes were simultaneously integrated into yeast chromosomes based on the M2S technique, and the yield of friedelin in recombinant strain GH1 was approximately 7-fold higher than that in the wild-type strain BY4741. Then, a transcriptional regulator and three genes that impact the triterpenoid synthesis pathway and the genes of the competitive pathway were knocked out by CRISPR/Cas9 technology, and the yield of friedelin in the quadruple mutant GQ1 (