Screening of astaxanthin-overproducing Xanthophyllomyces dendrorhous strains via iterative ARTP mutagenesis and cell sorting by flow cytometry.

AIMS: The astaxanthin-producing yeast Xanthophyllomyces dendrorhous is widely used in aquaculture. Due to the production of carotenoid, this yeast shows visible color; however, high-throughput approaches for identification of astaxanthin-overproducing strains remain rare. METHODS AND RESULTS: This study verified an effective approach to identify astaxanthin-overproducing mutants of X. dendrorhous by flow cytometry (FCM) and cell sorting. First, the mutant libraries were generated by atmospheric and room-temperature plasma (ARTP) mutagenesis. Second, a highly direct correlation between the concentrations of intracellular astaxanthin and the levels of emitting fluorescence was constructed by testing a variety of astaxanthin-contained populations via FCM and cell sorting. Third, iterative cell sorting efficiently improves the identification of astaxanthin-overproducing strains. Finally, two mutants producing 4.96 mg astaxanthin g-1 DCW (dry cell weight) and 5.30 mg astaxanthin g-1 DCW were obtained, which were 25.3% and 33.8% higher than that of the original strain, respectively. CONCLUSIONS: This study demonstrated that iterative ARTP mutagenesis along with cell sorting by FCM is effective for identifying astaxanthin-overproduction strains.

Integrating Transcriptomics and Proteomics to Characterize the Intestinal Responses to Cadmium Exposure Using a Piglet Model.

Cadmium (Cd) is a heavy metal element with a wide range of hazards and severe biotoxicity. Since Cd can be easily accumulated in the edible parts of plants, the exposure of humans to Cd is mainly through the intake of Cd-contaminated food. However, the intestinal responses to Cd exposure are not completely characterized. Herein, we simulated laboratory and environmental Cd exposure by feeding the piglets with CdCl2-added rice and Cd-contaminated rice (Cdcr) contained diet, as piglets show anatomical and physiological similarities to humans. Subsequent analysis of the metal element concentrations showed that exposure to the two types of Cd significantly increased Cd levels in piglets. After verifying the expression of major Cd transporters by Western blots, multi-omics further expanded the possible transporters of Cd and found Cd exposure causes wide alterations in the metabolism of piglets. Of significance, CdCl2 and Cdcr exhibited different body distribution and metabolic rewiring, and Cdcr had stronger carcinogenic and diabetes-inducing potential. Together, our results indicate that CdCl2 had a significant difference compared with Cdcr, which has important implications for a more intense study of Cd toxicity.

Comprehensive evaluation of factors influencing selenium fertilization biofortification.

BACKGROUND: Dietary selenium (Se) deficiency, stemming from low Se concentrations in agricultural products, threatens human health. While Se-containing fertilizers can enhance the Se content in crops, the key factors governing Se biofortification with Se fertilization remain unclear. RESULTS: This study constructed a global meta-analysis dataset based on field experiments comprising 364 entries on Se content in agricultural products and 271 entries on their yield. Random forest models and mixed effects meta-analyses revealed that plant types (i.e., cereals, vegetables, legumes, and forages) primarily influenced Se biofortification, with Se fertilization rates being the next significant factor. The random forest model, which included variables like plant types, Se fertilization rates, methods and types of Se application, initial soil conditions (including Se content, organic carbon content, and pH), soil types, mean annual precipitation, and temperature, explained 82.14% of the variation in Se content and 48.42% of the yield variation in agricultural products. For the same agricultural products, the increase in Se content decreased with higher rates of Se fertilization. The increase in Se content in their edible parts will be negligible for cereals, forages, legumes, and vegetable crops, when Se fertilization rates were 164, 103, 144, and 147 g Se ha-1, respectively. Conversely, while low Se fertilization rates enhanced yields, high rates led to a yield reduction, particularly in cereals. CONCLUSION: Our findings highlight the need for balanced and precise Se fertilization strategies to optimize Se biofortification benefits and minimize the risk of yield reduction. © 2024 Society of Chemical Industry.

Effects of different concentrations of coated nano zinc oxide material on fecal bacterial composition and intestinal barrier in weaned piglets.

BACKGROUND: Coated nano zinc oxide (Cnz) is a new feed or food additive, which is a potential replacement for a pharmacological dose level of ZnO. This study evaluated the positive effects of different concentrations of Cnz on the intestinal bacterial core, enterobacterial composition and mucosal barrier function in a pig model. RESULTS: Microbiota sequencing results showed that Cnz could significantly alter the intestinal microbiota composition and metabolism. Besides increasing the richness indices (ACE and Chao1), 10% Cnz could protect the intestinal mucosal barrier through increasing the expression of occludin and zonula occludens-1 in the small intestine, increase the abundance of Lachnospiraceae UCG-004 and decrease the abundance of Ruminococcus flavefaciens compared to high ZnO diet and 5% Cnz material. CONCLUSIONS: Cnz material at 10% supplementation is more effective than a level of 5% Cnz in increasing intestinal barrier through affecting gut microbiota. © 2020 Society of Chemical Industry.

Influence of supplemented coated-cysteamine on morphology, apoptosis and oxidative stress status of gastrointestinal tract.

BACKGROUND: Cysteamine was coated to cover its odor and maintain the stability. However, coated cysteamine (CC) has not been clearly evaluated for its effects on the gastrointestinal mucosa status. We hypothesize that the appropriate CC supplementation in diet impacts the stomach and intestinal mucosa variously through regulating the morphology, apoptosis, and oxidative stress status in model of pigs. RESULTS: The results showed that villus height increased (P < 0.05), and crypt depth decreased (P < 0.05) in the ileum when pigs were fed the diet with low cysteamine (LCS) compared with the control diet. The ileal lesion score in the LCS group was significantly (P < 0.01) lower than that in the control group, while the gastric lesion score in the CC group was significantly (P < 0.01) higher compared with that of the control group. It also showed that the activities of total superoxide dismutase (T-SOD) and diamine oxidase (DAO) were upregulated (P < 0.05) in the LCS group. In addition, Bax and caspase 3 immunore-activity increased (P < 0.01), and Bcl-2 immunoreactivity decreased (P < 0.01) in the gastric mucosa of pigs fed the diet with high cysteamine (HCS). The Bax and caspase 3 immunoreactivity decreased (P < 0.01), and Bcl-2 immunoreactivity increased (P < 0.01) in ileum mucosa of pigs fed the HCS diet. CONCLUSIONS: Although moderate dietary coated cysteamine showed positive effects on GI mucosal morphology, apoptosis, and oxidative stress status, the excess coated cysteamine may cause apoptosis leading to GI damage in pigs.

Dietary mulberry leaf powder affects growth performance, carcass traits and meat quality in finishing pigs.

This study was conducted to evaluate the effect of mulberry leaves as an alternative source of protein on growth performance, carcass traits and meat quality in finishing pigs. A total of 180 Xiangcun Black pigs were randomly assigned to five treatment groups with six pens of six pigs per pen. The pigs were provided with a basal diet or a diet contained 3%, 6%, 9% or 12% of mulberry leaf powder during a 50-day experiment period. The results showed that dietary mulberry leaf powder had no negative effect on growth performance in Xiangcun Black pigs, except in the 12% mulberry group, where final body weight and average daily gain decreased (p < .05) and feed to gain ratio of the pigs increased (p < .05). Dietary mulberry inclusion decreased (quadratic, p < .05) the back fat thickness, fibre mean cross-sectional area (CSA) in the longissimus dorsi (LD) muscle and mRNA expression levels of myosin heavy chain (MyHC) IIb in LD and biceps femoris (BF) muscles, while increased (linear or quadratic, p < .05) the plasma concentration of albumin, levels of crude protein (CP), inosine monophosphate (IMP) and several amino acids in muscle tissues. When compared with the other groups, the 9% mulberry diet increased (p < .05) loin-eye area and contents of CP and IMP in muscles, while decreased (p < .05) plasma activity of cholinesterase and concentrations of uric acid and urea. The 6% mulberry diet had the lowest fibre mean CSA and shear force and increased total fibre number of the LD muscle, when compared with the other groups. These results suggest that including mulberry in the diet at <12% is an effective feed crop to improve meat quality and the chemical composition of muscle without negatively affecting growth performance.

Galactose Derivative-Modified Nanoparticles for Efficient siRNA Delivery to Hepatocellular Carcinoma.

Successful siRNA therapy requires suitable delivery systems with targeting moieties such as small molecules, peptides, antibodies, or aptamers. Galactose (Gal) residues recognized by the asialoglycoprotein receptor (ASGPR) can serve as potent targeting moieties for hepatocellular carcinoma (HCC) cells. However, efficient targeting to HCC via galactose moieties rather than normal liver tissues in HCC patients remains a challenge. To achieve more efficient siRNA delivery in HCC, we synthesized various galactoside derivatives and investigated the siRNA delivery capability of nanoparticles modified with those galactoside derivatives. In this study, we assembled lipid/calcium/phosphate nanoparticles (LCP NPs) conjugated with eight types of galactoside derivatives and demonstrated that phenyl ß-d-galactoside-decorated LCP NPs (L4-LCP NPs) exhibited a superior siRNA delivery into HCC cells compared to normal hepatocytes. VEGF siRNAs delivered by L4-LCP NPs downregulated VEGF expression in HCC in vitro and in vivo and led to a potent antiangiogenic effect in the tumor microenvironment of a murine orthotopic HCC model. The efficient delivery of VEGF siRNA by L4-LCP NPs that resulted in significant tumor regression indicates that phenyl galactoside could be a promising HCC-targeting ligand for therapeutic siRNA delivery to treat liver cancer.

Transcriptomic analysis on responses of the liver and kidney of finishing pigs fed cadmium contaminated rice.

BACKGROUND: Cadmium (Cd) is a common harmful substance that has many deleterious effects on the liver and kidney. Most reports about Cd toxic studies focused on its inorganic status, whereas the toxicity of Cd in organic materials is less studied. Here, we performed RNA-seq to explore the influences of Cd contaminated rice on function of the liver and kidney of finishing pigs. RESULTS: The concentration of Cd in liver and kidney of pigs fed Cd contaminated rice increased by 4.00 and 2.94 times, respectively, compared to those in the control group. With transcriptomic analysis, approximately 4-6 × 107 clean reads were acquired. Five differently expressed genes (DEGs) were identified in the liver, and 12 DEGs in the kidney. SPHK2 was commonly down-regulated. No significantly enriched gene ontology (GO) terms were identified. By Kyoto encyclopaedia of genes and genomes (KEGG) enrichments, four pathways were identified in hepatic tissue, and five pathways in nephritic tissue. Intriguingly, two pathways (sphingolipid metabolism and VEGF signalling pathway) were altered both in the liver and kidney. CONCLUSION: Cd contaminated rice may cause liver and kidney damage and inflammation, or even lead to more severe harm to these tissues. © 2017 Society of Chemical Industry.

The effect of aspartate supplementation on the microbial composition and innate immunity on mice.

The study was conducted to investigate the changes of intestinal microbiota composition and innate immunity with different dietary dosages of aspartate (Asp) supplementation. Thirty-six female ICR mice were divided randomly to four groups and thereafter fed the basal diets (controls) or those supplemented with additional 0.5, 1.0 and 2.0% aspartate. After 2 week feeding, microbial composition in ileum and feces, gene expression of pro-inflammatory cytokine, and innate immune factors in ileum were determined. The ratio of Firmicutes: Bacteroidetes in ileum and feces decreased in 0.5 and 1.0% Asp-supplemented groups, whereas this ratio increased in feces in 2.0% Asp-supplemented group. Meanwhile, the gene expression of IL-17 and IFN-γ in ileum decreased in 1.0% Asp-supplemented group; the gene expression in ileum of Muc2 decreased in 0.5 and 1.0% Asp-supplemented groups. Dietary supplementation with 2.0% Asp enhanced the expression of pIgR and Crp1 as compared to the other three groups. The results indicated that dietary 1.0% Asp supplementation lowers the ratio of Firmicutes:Bacteroidetes, which affects the innate immunity by decreasing the gene expression of IL-17, IFN-γ, and Muc2 in ileum.

Acute and sub-acute oral toxicological evaluations and mutagenicity of N-carbamylglutamate (NCG).

N-carbamylglutamate (NCG) is a metabolically stable analog of N-acetylglutamate that activates carbamyl phosphate synthase-1, a key arginine synthesis enzyme in enterocytes. It is a promising feed additive in swine in China. In this study, we assessed the acute and sub-acute toxicity of NCG in Sprague-Dawley (SD) rats. All rats survived until they were killed at a scheduled time point. No adverse effects or mortality was observed following acute oral administration of 5000 mg/kg NCG to SD rats. No biologically significant or test substance-related differences were observed in body weights, feed consumption, clinical signs, a functional observational battery, organ weights, histopathology, ophthalmology, hematology, coagulation, and clinical chemistry parameters in any of the treatment groups in sub-acute doses of NCG at target concentrations corresponding to 500, 2000, and 3000 mg/kg/day for 28 days neither. In addition, no evidence of mutagenicity or genotoxicity was found, either in vitro in bacterial reverse mutation assay or in vivo in mice bone marrow micronucleus assay and sperm shape abnormality assay. On the basis of our findings, we conclude that NCG is a non-toxic substance with no genotoxicity.

Improving Efficiency: Automatic Intelligent Weighing System as a Replacement for Manual Pig Weighing.

To verify the accuracy of AIWS, we weighed 106 pen growing-finishing pigs' weights using both the manual and AIWS methods, respectively. Accuracy was evaluated based on the values of MAE, MAPE, and RMSE. In the growth experiment, manual weighing was conducted every two weeks and AIWS predicted weight data was recorded daily, followed by fitting the growth curves. The results showed that MAE, MAPE, and RMSE values for 60 to 120 kg pigs were 3.48 kg, 3.71%, and 4.43 kg, respectively. The correlation coefficient r between the AIWS and manual method was 0.9410, and R2 was 0.8854. The two were extremely significant correlations (p < 0.001). In growth curve fitting, the AIWS method has lower AIC and BIC values than the manual method. The Logistic model by AIWS was the best-fit model. The age and body weight at the inflection point of the best-fit model were 164.46 d and 93.45 kg, respectively. The maximum growth rate was 831.66 g/d. In summary, AIWS can accurately predict pigs' body weights in actual production and has a better fitting effect on the growth curves of growing-finishing pigs. This study suggested that it was feasible for AIWS to replace manual weighing to measure the weight of 50 to 120 kg live pigs in large-scale farming.

Warming Leads to Changes in Soil Organic Carbon Molecules Due to Decreased Mineral Protection.

Climate change affects the content and composition of soil organic carbon (SOC). However, warming-induced changes in the SOC compounds remain unknown. Using nuclear magnetic resonance spectroscopy, molecular mixing models, and Fourier transform ion cyclotron resonance mass spectrometry, we analyzed the variations and relationships in molecular compounds in Mollisol with 10-56 g C kg-1 soil-1 by translocating soils under six climate regimes. We found that increased temperature and precipitation were negatively correlated with carbohydrate versus lipid and lignin versus protein. The former was consistent across soils with varying SOC contents, but the latter decreased as the SOC content increased. The carbohydrate-lipid correlations were related to dithionite-citrate-extractable Fe, while the lignin-protein correlations were linked to changes in moisture and pyrophosphate-extractable Fe/Al. Our findings indicate that the reduction in the mineral protection of SOC is associated with molecular alterations in SOC under warming conditions.

Dietary supplementation with proline confers a positive effect in both porcine circovirus-infected pregnant and non-pregnant mice.

Porcine circovirus type 2 (PCV2) is associated with various diseases that impose a significant economic burden on the swine industry. We hypothesised that nutritional supplementation with proline to enhance the immune response might be a useful prophylactic measure against PCV2 infection. To test this hypothesis, in the present study, we measured clinical data, including blood parameters, serum cytokine profile, PCV2 virus load in organs and serum, and microscopic lesions in the lung, liver and spleen, in both PCV2-infected pregnant and non-pregnant mice. Dietary supplementation with proline had no effect (P>0·05) on abortion rates in PCV2-infected pregnant mice, although a numerically lower abortion rate (22·2 v. 44·4%) was observed compared with the control. Dietary supplementation with proline significantly increased serum C-reactive protein levels (P= 0·03) in PCV2-infected pregnant mice, and increased serum TNF-α levels (P= 0·01), leucocytes (P< 0·05), lymphocytes (P< 0·05) and neutrophilic granulocytes (P< 0·05) in PCV2-infected non-pregnant mice. Meanwhile, dietary proline significantly (P< 0·05) decreased the PCV2 virus load in the lung. Furthermore, mice in the dietary proline group showed a significant (P< 0·01) decrease in microscopic lesion scores in the lung, liver and spleen compared with those in the alanine group. Collectively, dietary proline supplementation confers a functional role in PCV2-infected mice.

Effect of L-arginine on HSP70 expression in liver in weanling piglets.

BACKGROUND: This study was conducted to evaluate the effects of L-arginine (Arg) on photomicrographs and HSP70 expression in the liver of weanling piglets. Twelve healthy Landrace × Yorkshire piglets that had been weaned at 21 d (average body weight 5.56 ± 0.51 kg) were randomly divided into a control group and an Arg group (6 g/kg feed). At age 28 d, all of the piglets were slaughtered to obtain liver samples to determine HSP70 expression by real-time PCR, western blot and immunohistochemistry. RESULTS: The results showed that, compared to control piglets, treatment with Arg decreased inflammatory reactions caused by weaning. The immunohistochemical localization of HSP70 in liver revealed strong expression in the Arg group. Arg increased HSP70 mRNA and HSP70 expression in the liver (P < 0.05). CONCLUSIONS: These findings suggest that dietary supplementation with Arg could maintain liver health by inducing HSP70 expression in weanling piglets.

Prediction of available energy and amino acid digestibility of Chinese sorghum fed to growing-finishing pigs.

Two experiments were conducted to determine digestible energy (DE), metabolizable energy (ME), as well as the standardized ileal digestibility (SID) of crude protein (CP) and amino acids (AA) in 10 sorghum samples fed to pigs. In experiment 1, 22 crossbred barrows (Duroc × Yorkshire × Landrace, Initial body weight [BW]: 70.0 ±â€…1.8 kg) were selected and allotted to a replicated 11 × 3 incomplete Latin square design, including a basal diet and 10 sorghum energy diets and three consecutive periods. Each period had 7 d adaptation and 5 d total feces and urine collection. The DE and ME were determined by the total collection and the difference method. In experiment 2, 22 crossbred barrows (Duroc × Yorkshire × Landrace, Initial BW: 41.3 ±â€…1.2 kg) that had a T-cannula installed in the distal ileum were assigned to a replicated 11 × 3 incomplete Latin square design, including an N-free diet and 10 sorghum diets. Each period had 5 d adaptation and 2 d ileal digesta collection. The basal endogenous N losses were measured by the N-free diet method. All diets in experiment 2 were added 0.30% titanium dioxide as an indigestible marker for calculating the ileal CP and AA digestibility. On an as-fed basis, the DE and ME contents in sorghum were 3,410 kcal/kg (2,826 to 3,794 kcal/kg) and 3,379 kcal/kg (2,785 to 3,709 kcal/kg), respectively. The best-fit prediction equation for DE and ME were DE = 6,267.945 - (1,271.154 × % tannin) - (1,109.720 × % ash) (R2 = 0.803) and ME = 51.263 + (0.976 × DE) (R2 = 0.994), respectively. The SID of CP, Lys, Met, Thr, and Trp (SIDCP, SIDLys, SIDMet, SIDThr, and SIDTrp) in 10 sorghum samples were 78.48% (69.56% to 84.23%), 74.27% (61.11% to 90.60%), 92.07% (85.16% to 95.40%), 75.46% (66.39% to 80.80%) and 87.99% (84.21% to 92.37%), respectively. The best prediction equations for SID of CP and the first four limiting AAs were as following: SIDCP = 93.404 - (21.026 × % tannin) (R2 = 0.593), SIDCP = 42.922 - (4.011 × % EE) + (151.774 × % Met) (R2 = 0.696), SIDLys = 129.947 - (670.760 × % Trp) (R2 = 0.821), SIDMet = 111.347 - (232.298 × % Trp) (R2 = 0.647), SIDThr = 55.187 + (3.851 × % ADF) (R2 = 0.609) and SIDTrp = 95.676 - (10.824 × % tannin) (R2 = 0.523), respectively. Overall, tannin and ash are the first and second predictors of DE and ME values of sorghum, respectively, and the tannin, EE, Trp, ash, CF, and ADF can be used as the key predictors for SID of CP and first four limiting AAs.

In this manuscript, we selected and analyzed the chemical composition of 10 different varieties of Chinese sorghum. Two digestion and metabolism experiments were conducted to measure the concentrations of available energy (digestible energy and metabolizable energy) and ileal amino acid digestibility (apparent or standardized ileal amino acid digestibility) in sorghums fed to pigs. The results showed as following: 1) The available energy values of 10 sorghum samples varied greatly, and the ileal digestibility of some amino acids also differed. 2) The available energy value and the ileal digestibility of crude protein and amino acids can be predicted by the contents of chemical components in sorghum, such as tannin (a substance in sorghum that can bind to biological macromolecules like proteins), ash, tryptophan, and fiber composition, etc. The above findings enrich the ingredient database and assist for feed enterprises to make precise formulations when using the Chinese sorghum.

Poor accuracy of single serological IgM tests in children with suspected acute Mycoplasma pneumoniae infection in Guangzhou, China.

Introduction. Early and accurate diagnosis of Mycoplasma pneumoniae (MP) infection of children with pneumonia is at the core of treatment in clinical practice.Gap Statement. Serological immunoglobulin M (IgM) tests for MP infection of children in south China have been rarely described.Aim. To assess the diagnostic performance and clinical application of serodiagnosis of MP infection in paediatric pneumonia patients.Methodology. Serum samples from 144 children diagnosed with MP pneumonia were subjected to a particle agglutination (PA)-based IgM assay. Meanwhile, we used an established suspension array as the reference standard method for the detection of MP DNA in bronchoalveolar lavage fluid (BALF) from all patients to assess the reliability of serological assays.Results. When running immunological testing in single serum samples, 80.6 %(79/98) of cases were diagnosed with MP infection, whereas only 55 (56.1 %) cases were positive in MP DNA analysis. Furthermore, single serum tests for IgM during acute MP infection resulted in 85.5 % (47/55) sensitivity and 25.6 % (11/43) specificity. Nevertheless, immunological testing and MP DNA analysis yielded the same results when paired sera were available for MP IgM antibody testing.Conclusion. Paired serological IgM assays are necessary for the determination of an acute MP infection, whereas single serological IgM testing is unreliable. Moreover, even a short interval of two MP serological tests works well.

Chemerin regulates proliferation and differentiation of myoblast cells via ERK1/2 and mTOR signaling pathways.

Obesity in human is an alarming major public health crisis worldwide and insulin resistance is a hallmark of it. The negative cross-talk between skeletal muscle and adipose tissue through adipokines is now accepted as one of the leading cause of insulin resistance. Chemerin is a novel adipokine previously reported to induce insulin resistance in primary human skeletal muscle cells. To investigate the role of chemerin in myogenesis, C2C12 cells were used and treated with chemerin in proliferation and differentiation stages. Our results showed that chemerin promoted proliferation and suppressed differentiation of C2C12 cells through extracellular-signal regulated kinase-1/2 (ERK1/2) and mammalian target of rapamycin (mTOR) signaling pathways, and these two pathways were interacted with each other in C2C12 cells treated with chemerin. It is concluded from this in vitro study that chemerin which expression is increased during myoblast differentiation appears to be able, likely in an autocrine/paracrine manner, to increase myoblast proliferation and decrease myoblast differentiation.

GATA transcription factor WC2 regulates the biosynthesis of astaxanthin in yeast Xanthophyllomyces dendrorhous.

Astaxanthin is a type of carotenoid widely used as powerful antioxidant and colourant in aquaculture and the poultry industry. Production of astaxanthin by yeast Xanthophyllomyces dendrorhous has attracted increasing attention due to high cell density and low requirements of water and land compared to photoautotrophic algae. Currently, the regulatory mechanisms of astaxanthin synthesis in X. dendrorhous remain obscure. In this study, we obtained a yellow X. dendrorhous mutant by Atmospheric and Room Temperature Plasma (ARTP) mutagenesis and sequenced its genome. We then identified a putative GATA transcription factor, white collar 2 (XdWC2), from the comparative genome data and verified that disruption of the XdWC2 gene resulted in a similar carotenoid profile to that of the ARTP mutant. Furthermore, transcriptomic analysis and yeast one-hybrid (Y1H) assay showed that XdWC2 regulated the expression of phytoene desaturase gene CrtI and astaxanthin synthase gene CrtS. The yeast two-hybrid (Y2H) assay demonstrated that XdWC2 interacted with white collar 1 (XdWC1) forming a heterodimer WC complex (WCC) to regulate the expression of CrtI and CrtS. Increase of the transcriptional levels of XdWC2 or CrtS in the wild-type strain did not largely modify the carotenoid profile, indicating translational and/or post-translational regulations involved in the biosynthesis of astaxanthin. Overexpression of CrtI in both the wild-type strain and the XdWC2-disrupted strain apparently improved the production of monocyclic carotenoid 3-hydroxy-3', 4'-didehydro-ß, ψ-carotene-4-one (HDCO) rather than ß-carotene and astaxanthin. The regulation of carotenoid biosynthesis by XdWC2 presented here provides the foundation for further understanding the global regulation of astaxanthin biosynthesis and guides the construction of astaxanthin over-producing strains.

Comprehensive Analysis of CRISPR-Cas9 Editing Outcomes in Yeast Xanthophyllomyces dendrorhous.

DNA repair after Cas9 cutting can result in deletions/insertions, genomic rearrangements, and rare nucleotide substitutions. However, most work has only focused on deletions/insertions resulting from repair after CRISPR-Cas9 action. Here, we comprehensively analyzed the editing outcomes induced by CRISPR-Cas9 treatment in yeast Xanthophyllomyces dendrorhous by Sanger and Illumina sequencing and identified diverse DNA repair patterns, including DNA deletions, interchromosomal translocations, and on-target nucleotide substitutions (point mutations). Some deletions were observed repeatedly, and others, especially large deletions, varied in size. Genome sequencing and structural variation analysis showed that the interchromosomal translocations happened between Cas9 target sites and the endogenous ADH4 promoter. In contrast to previous studies, analysis revealed that the on-target point mutations were not random. Importantly, these point mutations showed strong sequence dependence that is not consistent with previous work in Hela cells, where CRISPR-mediated substitutions were found to lack sequence dependence and conversion preferences. Finally, we found that the non-homologous end joining components Ku70, Ku80, Mre11, or RAD50, and the overlapping roles of non-essential DNA polymerases were necessary for the production of both point mutations and deletions. This work expands our knowledge of CRISPR-Cas9 mediated DNA repair.

Comparative and evolutionary analysis of RIP kinases in immune responses.

The group of receptor-interacting protein (RIP) kinases has seven members (RIPK1-7), with one homologous kinase domain but distinct non-kinase regions. Although RIPK1-3 have emerged as key modulators of inflammation and cell death, few studies have connected RIPK4-7 to immune responses. The divergence in domain structures and paralogue information in the Ensembl database have raised question about the phylogeny of RIPK1-7. In this study, phylogenetic trees of RIPK1-7 and paralogues constructed using full-length amino acid sequences or Kinase domain demonstrate that RIPK6 and RIPK7 are distinct from RIPK1-5 and paralogues shown in the Ensembl database are inaccurate. Comparative and evolutionary analyses were subsequently performed to gain new clues about the potential functions of RIPK3-7. RIPK3 gene loss in birds and animals that undergo torpor, a common physiological phenomenon in cold environments, implies that RIPK3 may be involved in ischemia-reperfusion injury and/or high metabolic rate. The negligible expression of RIPK4 and RIPK5 in immune cells is likely responsible for the lack of studies on the direct role of these members in immunity; RIPK6 and RIPK7 are conserved among plants, invertebrates and vertebrates, and dominantly expressed in innate immune cells, indicating their roles in innate immunity. Overall, our results provide insights into the multifaceted and conserved biochemical functions of RIP kinases.