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Uncertainty exists about whether lowering systolic blood pressure to less than 120 mm Hg is superior to that of less than 140 mm Hg, particularly in patients with diabetes and patients with previous stroke.

Suancai is a traditional fermented vegetable widely consumed in Northeast China. In this study, different prebiotics were used to improve the quality of suancai. Four prebiotics (inulin (INU), xylooligosaccharide (XOS), galactooligosaccharide (GOS), and stachyose (STA)) were shown to reduce the pH value and increase the content of total titratable acidity (TTA) in suancai, while the contents of most organic acids were also increased. The addition of prebiotics had significant effects on the bacterial microbiota during the suancai fermentation process. All prebiotics were shown to contribute to the growth of Lactobacillus. The suancai sample with fructooligosaccharides (FOS) had the highest relative abundance of Lactobacillus. Besides, INU and XOS could increase the abundance of Weissella. To evaluate the quality of suancai fermented with prebiotics, profiles of volatile flavor compounds (VOCs) and free amino acids (FAA) were analyzed. The prebiotics affected the VOCs and FAA profiles via transforming the bacterial microbiota. In addition, the addition of prebiotics also changed the taste profiles of the suancai samples. This study is among the first attempts to reveal the effects of different prebiotics on suancai fermentation, and the findings provide a foundation to develop new ways for improving the quality of suancai.

Increasing the copy number of peptides is an effective method to genetically engineer recombinant expression and obtain umami peptides in large quantities. However, the umami taste value of multicopy number umami peptides is lower than the single ones, thus limiting the industrial application of recombinantly expressed umami peptides. With aims to solve this problem, modification of an umami beefy meaty peptide (BMP) with trypsin hydrolysis sites was carried out via homology modeling and molecular docking in this study. A total of 1286 modified peptide sequences were created and molecularly simulated for docking with the homology modeling-constructed umami receptor (T1R1/T1R3), and 837 peptides were found to be better docked than the BMP. Afterward, the MLSEDEGK peptide with the highest docking score was synthesized. And umami taste evaluation results demonstrated that this modified peptide was close to that of monosodium glutamate (MSG) and BMP, as confirmed by electronic tongue and sensory evaluation (umami value: 8.1 ± 0.2 for BMP; 8.2 ± 0.3 for MLSEDEGK peptide). Meanwhile, mock trypsin digestion of eight copies of MLSEDEGK peptide results showed that the introduced digestion sites were effective. Therefore, the novel modified BMP in this study has the potential for large-scale production by genetic engineering.

Suitable selection of endogenous controls is necessity for obtaining reliable qRT-PCR results. However, insufficient attention has been paid to the selection of endogenous controls for normalization in different stresses in plant, especially Larix olgensis. In this study, we screened candidate internal reference genes for miRNA expression normalization after subjecting Larix olgensisPEG-induced drought, NaHCO3, or NaCl stress. Needles samples were collected at 0, 12, 24, 48 and 96 h after treatment. Four common housekeeping genes and 11 mature miRNAs that were found to be stably expressed in small RNA sequencing data of Larix olgensiswere selected as candidate internal reference genes. Their expression levels were assessed by qRT-PCR. GeNorm and NormFinder were used to analyse the stability of gene expression from the candidate internal reference genes. Finally, the expression of some miRNAs was analysed after stress. The results showed that miRNAs exhibited better expression stability compared to the analysed ncRNAs. Under PEG, NaHCO3, or NaCl stress, miR83321, miR43169, and miR74940 were identified as the most suitable internal reference genes, respectively. Under PEG stress, the expression of most miRNAs reached its highest level at 12 h, showing a trend of first increasing expression and then decreasing. Similarly, under NaHCO3and NaCl stress, the expression of most miRNAs reached its maximum levels at 48 h and 12 h, respectively. This indicates that most miRNAs more quickly respond to PEG and NaCl stress than NaHCO3stress.

In order to explore novel producing strategies for enhancing flavor compounds profile and quality of strawberry wine, we studied the effect of the sequential fermentation of two selected indigenous non-Saccharomycesyeasts (Hanseniaspora uvarumPGR9 and Kurtzmaniella quercitrusaPGT1) and Saccharomyces cerevisiaeCGMCC 2.3854 on the fermentation kinetics, physicochemical characteristics, organic acids, antioxidant activity, volatile compositions, and sensory properties. Results showed that the ethanol content produced by the fermentation of Kq-Scwas significantly lower than that of Scpure fermentation. In addition, the total volatile compounds concentration produced by the fermentation of Hu-Sc(16.2 ± 3.6 mg/L) and Kq-Sc(19.5 ± 3.1 mg/L) were significantly higher than that of Scpure fermentation (13.8 ± 2.0 mg/L). In particular, the Kq-Scstrawberry wine presented the highest number of relative odor-active compounds (rOAV ≥1). Moreover, strawberry wine fermented by Hu+Kq-Scshowed the highest ABTS free radical scavenging ability and the highest overall sensory qualities. The results indicated that the K. quercitrusaPGT1 strain could be an alternative yeast for producing strawberry wine, and the sequential fermentations of Kq-Sc and Hu+Kq-Scwere effective strategies for improving the flavor and quality of strawberry wine.

Peptide ferrous chelate is a new type of biological iron, which has the advantages of high absorption rate, safety and no digestive tract irritation. In this study, microbial fermentation was used for the first time to prepare iron-chelating peptides. Forty-eight strains were applied to ferment scallop skirts. A dominating strain was eliminated by using the iron binding capacity of the scallop skirt hydrolysate as the screening index. The scallop skirt hydrolysate fermented by Bacillus subtilisM17-b7 showed excellent iron-chelating capacity (96.20% ± 0.03%). Meanwhile, the structure, morphology and bioactivity of fermented scallop skirt hydrolysate (FSH) and fermented scallop skirt hydrolysate-iron (FSH–Fe) were measured. Based on the amino acid composition and spectral analysis, it was confirmed that the significant amino acids contains carboxyl, carbonyl, and amino groups. The result of scanning electron microscope indicated that the surface of FSH became porous and the particles were bigger after chelating with ferrous. Moreover, the peptide-ferrous chelate was more stable in salt solutions and simulated gastric juice compared with ferrous sulfate. It is also noteworthy that the preparation of ferrous chelate peptides by fermentation is cost-effective compared with enzymatic hydrolysis. Therefore, this study provides theoretical support for the use of fermentation as a promising tool to produce peptide-ferrous chelate.

Natto, a nutritious fermented soybean product, is known for its health benefits. Modern production methods use Bacillus subtilisas the starter culture to ensure consistent quality. However, this method overlooks the potential positive effects of other microbial species present in traditionally, naturally fermented natto. This study explored the effects of ARTP-induced mutants of B. subtilisvar. nattoand several strains of lactic acid bacteria (LAB) on natto quality. The results showed that these two types of bacteria have complementary roles in fermentation. B. subtilisvar. natto, with enhanced extracellular protease activity, promotes the hydrolysis of soy protein and enhances the product's ACE inhibitory and fibrinolytic activities. Meanwhile, two strains of Lactiplantibacillus plantarumreduced the content of total volatile basic nitrogen (TVB-N) while increasing the content of folic acid in the final product. In addition, B. subtilisvar. nattoand LAB produce different profiles of volatile compounds (VOCs). Subsequently, B. subtilisvar. nattoand Lpb. plantarumwere used to co-ferment natto in different ratios. The results showed that the two types of bacteria could co-colonize and enhance fermentation quality through their respective strengths. Specifically, Lpb. plantarumstrains co-metabolized with B. subtilisvar. nattoto elevate the content of pyrazines, characteristic VOCs in natto. The optimal quality of natto was achieved when Lpb. plantarumHIS6, Lpb. plantarumLP3 and B. subtilisvar. nattoY15-11 were combined in a 2:1:2 ratio. This study not only identified effective combinations of starter cultures with complementary benefits but also elucidated the metabolic pathways of key volatile compounds. Our findings also provide technical insights for improving natto quality, enhancing consumer acceptance, and supporting industrial production.

Wort, derived from malt saccharification, contains a myriad of vital nutrients, such as polyphenols, flavonoids, and sugars. Beer, a time-honored libation originating from malt fermentation, graces the palate with its rich flavor and purported health benefits. Unfortunately, due to its high alcohol content, consuming beer is considered inappropriate for pregnant women and children. Hence, there is a need to develop a low-alcohol or alcohol-free fermented beverage derived from wort. This research utilized water kefir grains to produce a low-alcohol fermented beverage from wort and examined the effects of water kefir grains fermentation on various attributes of the wort, including physicochemical properties, organic acid composition, sensory characteristics, volatile compounds, and microbial diversity. Remarkably, after fermentation, a new type of low-alcohol or alcohol-free wort beverage is produced. Moreover, the fermentation process generated numerous volatile components that enhanced the sensory profile of the beverage. The findings presented in this study serve as a valuable guide for the development of innovative low-alcohol or non-alcohol fermented beverages from wort.

Kiwifruit, a climacteric-fruit susceptible to rapid deterioration, attains considerable value upon transformation into kiwifruit wine, thereby increasing its added value. However, a noteworthy challenge arises from its high pectin content, which negatively affects byproduct quality, necessitating pectinase treatment during processing. Commercial pectinases exhibit heightened pectin esterase activity, giving rise to safety apprehensions due to augmented methanol content during kiwifruit pectin hydrolysis. In this study, Bacillus velezensisW17-6 was screened for its high pectinase activity coupled with low pectin esterase activity. The resultant crude pectinase named PBV was acquired through fed-batch-fermentation and partial purification. Subsequently, PBV was deployed in the fermentation of Xuxiang kiwifruit wine, and its impact on overall quality was assessed through diverse analyses. The results indicated that Xuxiang kiwifruit wine with PBV treatment outperformed counterparts treated with commercial pectinases. PBV treatment not only yielded superior clarity (83.75%±0.47%) and juice yield (81.60%±0.47%) but also increased the total flavonoids content in Xuxiang kiwifruit wine by 1.2 times compared to commercial pectinase. Notably, the methanol content of PBV-treated Xuxiang kiwifruit wine was significantly lower at 36.13±1.83 mg/L than that of wines treated with two commercial pectinases. PBV-treated kiwifruit wine also exhibited elevated concentrations of volatile compounds such as phenethyl alcohol, ethyl undecanoate, lauric aldehyde, isobutyric acid, and myrcene. Impressively, PBV-treated kiwifruit wine surpassed other wines in both electronic and artificial sensory analyses. In summary, PBV exhibits substantial potential in the production of low-methanol, high-quality kiwifruit wine, enhancing the flavor and nutritional value while minimizing methanol production and improving the safety profile.

In this study, a Petri-dish-based double-layer high-throughput screening method was established to improve glucose tolerance of β-glucosidase Bgl15. Two beneficial mutations were identified, and the joint mutant 2R1 improved the half-maximal inhibitory concentration of glucose from 0.04 to 2.1 M. The crystal structure of 2R1 was subsequently determined at 2.7 Å. Structure analysis revealed that enhancement of glucose tolerance may be due to improved transglycosylation activity made possible by a hydrophobic binding site for glucose as an acceptor and more stringent control of a putative water channel. To further ameliorate the application potential of the enzyme, it was engineered to increase the half-life at 50 °C from 0.8 h (Bgl15) to 180 h (mutant 5R1). Furthermore, supplementation of 5R1 to the cellulase cocktail significantly improved glucose production from pretreated sugar cane bagasse by 38%. Consequently, this study provided an efficient approach to enhance glucose tolerance and generated a promising catalyst for cellulose saccharification.

There are ongoing interests in improving the galactooligosaccharide (GOS) synthesis efficiency of β-galactosidase by protein engineering. In this study, an intelligent double-hydrophobic amino acid scanning strategy was proposed and employed to target nine residues forming the glycon-binding site (−1 subsite) of β-galactosidase Bgal1-3. Two mutants C510V and H512I with significantly improved GOS synthesis efficiency were obtained. When 40% (w/v) lactose was used as a substrate, Bgal1-3 reached a maximum GOS yield of 45.3% at 16 h, while the mutants reached higher yields in a much shorter time (59.1% at 10 h for C510V, 51.5% at 2 h for H512I). When skim milk was treated with these enzymes, more GOS was produced (19.9 g/L for C510V, 12.7 g/L for H512I) than that for Bgal1-3 (10.3 g/L) at a lactose conversion of 90%. These results validated hydrophobicity scanning as an efficient method to engineer β-galactosidases into promising catalysts for the preparation of GOS and GOS-enriched milk.

ABSTRACTChromosomal duplication is targeted by various chemotherapeutic agents for the treatment of cancer. However, there is no specific inhibitor of DNA polymerases that is viable for cancer management. Through structure-based in silicoscreening of the ZINC database, we identified a specific inhibitor of DNA polymerase δ. The discovered inhibitor, Zelpolib, is projected to bind to the active site of Pol δ when it is actively engaged in DNA replication through interactions with DNA template and primer. Zelpolib shows robust inhibition of Pol δ activity in reconstituted DNA replication assays. Under cellular conditions, Zelpolib is taken up readily by cancer cells and inhibits DNA replication in assays to assess global DNA synthesis or single-molecule bases by DNA fiber fluorography. In addition, we show that Zelpolib displays superior antiproliferative properties to methotrexate, 5-flourouracil, and cisplatin in triple-negative breast cancer cell line, pancreatic cancer cell line and platinum-resistant pancreatic cancer cell line. Pol δ is not only involved in DNA replication, it is also a key component in many DNA repair pathways. Pol δ is the key enzyme responsible for D-loop extension during homologous recombination. Indeed, Zelpolib shows robust inhibition of homologous recombination repair of DNA double-strand breaks and induces “BRCAness” in HR-proficient cancer cells and enhances their sensitivity to PARP inhibitors.

ObjectiveThis study aimed to compare the hemodynamic differences among no sac (NOS), porous media (POM) and finite element analysis (FEA) models to investigate the recurrence-related risks for coiled intracranial aneurysms (IAs).MethodsThe study enrolled 10 patients with 11 IAs who received simple coiling treatment and hemodynamic simulations were performed for all IAs using the above three models. Velocity, wall shear stress (WSS) and residual flow volume (RFV) were calculated and compared in order to assess the model differences for both aneurysm sac and parent vessel regions.ResultsFor parent artery regions, all three models produced similar flow patterns and quantification analysis did not indicate differences in velocity and WSS (p>0.05). For aneurysm sac regions, the FEA model resulted in higher sac-maximized (0.18 m/s vs 0.06 m/s) and sac-averaged velocity (0.013 m/s vs 0.007 m/s), and higher sac-averaged (0.55 Pa vs 0.36 Pa, p=0.006) and sac-maximized WSS (12.1 Pa vs 6.6 Pa) than the POM model. The differences in RFV between the POM and FEA models under 11 different isovelocity thresholds (0.0001 m/s, 0.001 m/s, 0.002 m/s, 0.005 m/s, 0.01 m/s, 0.02 m/s, 0.05 m/s, 0.1 m/s, 0.2 m/s, 0.5 m/s, and 1 m/s) showed that the POM RFV was generally larger than those of the FEA model.ConclusionsCompared with the FEA model, the POM model provides a lower velocity and WSS and higher RFV for the aneurysm sac, which could lead to incorrect estimates of the recurrent risk of coiled IAs under high packing density.

The development of ultra-fast response semiconductor gas sensors for high-accuracy detection of trace volatile organic compounds in human exhaled breath still remains a challenge. Herein, we propose a novel self-assembly synthesis concept for preparing intricate three-dimensional (3D) opal porous (OP) SnO2–ZnO hollow microspheres (HM), by employing sulfonated polystyrene (S-PS) spheres template-assisted ultrasonic spray pyrolysis. The high gas accessibility of the unique opal hollow structures resulted in the existence of 3D interconnection and bimodal (mesoscale and macroscale) pores, and the n–n heterojunction-induced change in oxygen adsorption. The 3D OP SnO2–ZnO HM sensor exhibited high response and ultra-fast dynamic process (response time ∼4 s and recovery time ∼17 s) to 1.8 ppm acetone under highly humid ambient condition (98% relative humidity), and it could rapidly identify the states of the exhaled breath of healthy people and simulated diabetics. In addition, the rational structure design of the 3D OP SnO2HM enables the ultra-fast detection (within 1 s) of ethanol in simulation drunk driving testing. Our results obtained in this work provided not only a facile self-assembly approach to fabricate metal oxides with 3D OP HM structures but also a new methodology for achieving noninvasive real-time exhaled breath detection.

Ergothioneine (EGT) is a natural sulfur-containing amino acid with potent antioxidant and cytoprotective activities. In this study, the oxidation tolerance of 23 different Rhodotorulastrains were screened with hydrogen peroxide (H2O2) as the inhibitory condition. By measuring their carotenoid and EGT production and correlation analysis with H2O2tolerance, the results showed that the carotenoid synthesis capacity has no effect on H2O2tolerance, in contrast to a strong positive correlation between EGT yield and H2O2tolerance of Rhodotorulaspecies. In addition, a high EGT-producing strain R. mucilaginosaDL-X01 was screened with the yield of 38.48 ± 2.71 mg/L in a shaking flask fermentation. To the best of our knowledge, this is the highest EGT yield ever reported in non-recombinant strains. Growth kinetic data showed that DL-X01 had a longer lag phase in the presence of exogenous oxidant, but a shorter generation time and higher specific growth rate after entering the exponential phase. The results of this study provided a potential simple and rapid method for screening high EGT-producing strains, which could promote the efficient production of EGT by microorganisms and further development of the red yeast in the food and biotechnology industries.

The design of semiconductor metal oxides (SMOs) with well-ordered porous structure has attracted tremendous attention owing to their larger specific surface area. Herein, three-dimensional inverse opal In2O3microspheres (3D-IO In2O3MSs) were fabricated through one-step ultrasonic spray pyrolysis (USP) which employed self-assembly sulfonated polystyrene (S-PS) spheres as a sacrificial template. The spherical pores observed in the 3D-IO In2O3MSs had diameters of about 4 and 80 nm. Subsequently, the catalytic palladium oxide nanoparticles (PdO NPs) were loaded on 3D-IO In2O3MSs via a simple impregnation method, and their gas sensing properties were investigated. In a comparison with pristine 3D-IO In2O3MSs, the 3D-IO PdO@In2O3MSs exhibited a 3.9 times higher response (Rair/Rgas= 50.9) to 100 ppm acetone at 250 °C and a good acetone selectivity. The detection limit for acetone could extend down to ppb level. Furthermore, the 3D-IO PdO@In2O3MSs-based sensor also possess good long-term stability. The extraordinary sensing performance can be attributed to the novel 3D periodic porous structure, highly three-dimensional interconnection, larger specific surface area, size-tunable (meso- and macroscale) bimodal pores, and PdO NP catalysts.

Kombucha is a kind of traditional fermented sugared tea rich in polyphenols. Although the health effects of tea polyphenols (TPPs) have been extensively studied, the biotransformation of the TPPs during kombucha fermentation was not fully elucidated. In this study, the polyphenol transformation capacity of microbial strains and the synthetic microbial communities during the tea broth fermentation were investigated. Firstly, six microbial species demonstrated different polyphenol transformation abilities: Acetobacter pasteurianus, Zygosaccharomyces bailiiand Debaryomyce hanseniicould increase the contents of epigallocatechin gallate (EGCG), the most abundant components of TPPs. Z. bailiiand Acetobacter xylinumcould enhance the concentration of TPPs with low molecular weight. Then, four synthetic microbial communities (SMC) consisting of different species were constructed to ferment the black tea broth. When different strains coexisted, their interaction could further enhance kombucha's phenols transformation and antioxidant activity. Specifically, the SMC, constructed by Z. bailii, L. plantarumand D. hansenii, could enhance the EGCG content significantly in tea broth and the total phenols and flavonoid contents. Meanwhile, the antioxidant activities of kombucha fermented by this SMC were found to be relatively high among different fermented tea broth groups. Besides, it is demonstrated that A. pasteurianuswas a strong competitor in the tea broth environment. This bacterium could decrease the number of viable yeast cells by producing more acid. This study demonstrated that the construction of suitable synthetic microbial communities would help reveal the phenolic metabolic characteristics of complex microbial communities and obtain customizable phenolic combinations for kombucha products.

A series of carbazole-based vinyl-benzoxazole derivatives have been synthesized in order to verify whether X-ray diffraction (XRD) simulation can give more information about intermolecular stacking in the gel phase. It was found that their gelation capabilities were strongly dependent on the length of the alkyl chain. The compounds with shorter alkyl chains have lower critical gelation concentrations (CGCs) in nonpolar alkane and alcohols with longer carbon chains. On the other hand, compounds with long alkyl chains presented small CGCs in polar methanol. Powder XRD structure solution gave more information about intermolecular stacking than the traditional way of analyzing diffraction peaks to derive approximate molecular stacking patterns. The results verified that gelators had a similar head-to-tail π-stacking between aromatic groups in gel phases although different slipping angles existed. Moreover, ordered stacking between the alkyl chains was also present.

Ergothioneine (EGT) is a natural histidine-derived compound with strong antioxidant activity. This research evaluated the potential of EGT to protect against liver injury induced by carbon tetrachloride (CCl4) both in vivoand in vitrofor the first time. Animal results showed that daily supplement of EGT (continuous gavage for 21 days) significantly reduced the levels of elevated serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), regulated inflammatory factor levels, and improved antioxidant enzyme activity in mice after CCl4treatment. In addition, cell results showed that exposure to CCl4causes significant oxidative stress in LO2 cells, which was mitigated after EGT pretreatment. Furthermore, EGT treatment reduced reactive oxygen species production and improved mitochondrial function compared with the model group, which was potentially mediated by the activation of the Nrf2/ARE pathway. These findings provided a scientific theoretical basis for the application of EGT in prevention of liver injury.

Cancer cells rely on the activation of telomerase or the alternative lengthening of telomeres (ALT) pathways for telomere maintenance and survival. ALT involves homologous recombination (HR)-dependent exchange and/or HR-associated synthesis of telomeric DNA. Utilizing proximity-dependent biotinylation (BioID), we sought to determine the proteome of telomeres in cancer cells that employ these distinct telomere elongation mechanisms. Our analysis reveals that multiple DNA repair networks converge at ALT telomeres. These include the specialized translesion DNA synthesis (TLS) proteins FANCJ-RAD18-PCNA and, most notably, DNA polymerase eta (Polη). We observe that the depletion of Polη leads to increased ALT activity and late DNA polymerase δ (Polδ)-dependent synthesis of telomeric DNA in mitosis. We propose that Polη fulfills an important role in managing replicative stress at ALT telomeres, maintaining telomere recombination at tolerable levels and stimulating DNA synthesis by Polδ.

ABSTRACTLipid droplets (LDs) are ubiquitous organelles that serve as a neutral lipid reservoir and a hub for lipid metabolism. Manipulating LD formation, evolution, and mobilization in oleaginous species may lead to the production of fatty acid-derived biofuels and chemicals. However, key factors regulating LD dynamics remain poorly characterized. Here we purified the LDs and identified LD-associated proteins from cells of the lipid-producing yeast Rhodosporidium toruloidescultured under nutrient-rich, nitrogen-limited, and phosphorus-limited conditions. The LD proteome consisted of 226 proteins, many of which are involved in lipid metabolism and LD formation and evolution. Further analysis of our previous comparative transcriptome and proteome data sets indicated that the transcription level of 85 genes and protein abundance of 77 proteins changed under nutrient-limited conditions. Such changes were highly relevant to lipid accumulation and partially confirmed by reverse transcription-quantitative PCR. We demonstrated that the major LD structure protein Ldp1 is an LD marker protein being upregulated in lipid-rich cells. When overexpressed in Saccharomyces cerevisiae, Ldp1 localized on the LD surface and facilitated giant LD formation, suggesting that Ldp1 plays an important role in controlling LD dynamics. Our results significantly advance the understanding of the molecular basis of lipid overproduction and storage in oleaginous yeasts and will be valuable for the development of superior lipid producers.

We recently reported that the p12 subunit of human DNA polymerase δ (Pol δ4) is degraded by CRL4Cdt2which regulates the licensing factor Cdt1 and p21WAF1during the G1to S transition. Presently, we performed multiparameter laser scanning cytometric analyses of changes in levels of p12, Cdt1 and p21WAF1, detected immunocytochemically in individual cells, vis-à-vis the initiation and completion of DNA replication. The latter was assessed by pulse-labeling A549 cells with the DNA precursor ethynyl-2′-deoxyribose (EdU). The loss of p12 preceded the initiation of DNA replication and essentially all cells incorporating EdU were p12 negative. Completion of DNA replication and transition to G2phase coincided with the re-appearance and rapid rise of p12 levels. Similar to p12 a decline of p21WAF1and Cdt1 was seen at the end of G1phase and all DNA replicating cells were p21WAF1and Cdt1 negative. The loss of p21WAF1preceded that of Cdt1 and p12 and the disappearance of the latter coincided with the onset of DNA replication. Loss of p12 leads to conversion of Pol δ4 to its trimeric form, Pol δ3, so that the results provide strong support to the notion that Pol δ3 is engaged in DNA replication during unperturbed progression through the S phase of cell cycle. Also assessed was a correlation between EdU incorporation, likely reflecting the rate of DNA replication in individual cells, and the level of expression of positive biomarkers of replication cyclin A, PCNA and Ki-67 in these cells. Of interest was the observation of stronger correlation between EdU incorporation and expression of PCNA (r = 0.73) than expression of cyclin A (r = 0.47) or Ki-67 (r = 0.47).

The objective of this study was to investigate the effect of papain on the physicochemical and microbiological properties, volatile compound profiles, and sensorial attributes of Chouguiyu. Papain was applied at a dose of 10, 50, 200, 400 U/g fish. Results showed that papain addition improved the release of free amino acids, essential amino acids, eicosapentaenoi acid (EPA, C20:5n3), and docosapentenoic acid (DPA, C22:5n6), respectively. Lower biogenic amines content, higher the volatile compound concentrations and relative abundance of Lactobacillales were also observed. Further results showed that the papain dose group decreased the fermentation time from 10 to 7 days while good organoleptic quality was remained, especially in the 200 U/g dose group. The redundancy analysis (RDA) showed a high correlation between Lactobacillus and the key volatile compounds in the 200 U/g and 400 U/g groups, which indicated that Lactobacillus could contribute to the fatty flavor. In summary, proper papain addition (i.e. 200 U/g or 400 U/g) could reduce biogenic amines contents and maintain acceptable sensorial properties of Chouguiyu in a shorter fermentation time.

DNA polymerase δ (Pol δ) is a key enzyme in eukaryotic DNA replication. Human Pol δ is a heterotetramer whose p12 subunit is degraded in response to DNA damage, leading to the in vivo conversion of Pol δ4 to Pol δ3. Two E3 ubiquitin ligases, RNF8 and CRL4Cdt2, participate in the DNA damage-induced degradation of p12. We discuss how these E3 ligases integrate the formation of Pol δ3 and ubiquitinated PCNA for DNA repair processes. CRL4Cdt2partially degrades p12 during normal cell cycle progression, thereby generating Pol δ3 during S phase. This novel finding extends the current view of the role of Pol δ3 in DNA repair and leads to the hypothesis that it participates in DNA replication. The coordinated regulation of licensing factors and Pol δ3 by CRL4Cdt2now opens new avenues for control of DNA replication. A parallel study of Pol δ4 and Pol δ3 in Okazaki fragment processing provides evidence for a role of Pol δ3 in DNA replication. We discuss several new perspectives of the role of the 2 forms of Pol δ in DNA replication and repair, as well the significance of the integration of p12 regulation in DNA repair and cell cycle progression.

PDIP38 (polymerase delta interacting protein 38) was originally discovered as a protein that interacts with DNA polymerase δ and PCNA. PDIP38 is present in multiple intracellular locations and is a multifunctional protein that has been implicated in several diverse cellular functions. We investigated the nuclear localization of PDIP38 in order to gain insights to its response to UV damage. PDIP38 was found to form distinct nuclear foci in response to UV irradiation in several cell lines, including HeLa S3 and A549 cells. However, these foci were not those associated with UV repair foci. Using various markers for different nuclear subcompartments, the UV-induced PDIP38 foci were identified as spliceosomes/nuclear speckles, the storage and assembly sites for mRNA splicing factors. To assess the role of PDIP38 in the regulation of splicing events, the effects of PDIP38 depletion on the UV-induced alternate splicing of MDM2 transcripts were examined by nested RT-PCR. Alternatively spliced MDM2 products were induced by UV treatment but were greatly reduced in cells expressing shRNA targeting PDIP38. These findings indicate that upon UV-induced DNA damage, PDIP38 is translocated to spliceosomes and contributes to the UV-induced alternative splicing of MDM2 transcripts. Similar results were obtained when cells were subjected to transcriptional stresses with actinomycin D or α-amanitin. Taken together, these studies show that PDIP38 is a protein regulated in a dynamic manner in response to genotoxic stress, as evidenced by its translocation to the spliceosomes. Moreover, PDIP38 is required for the induction of the alternative splicing of MDM2 in response to UV irradiation.

Autophagy plays an important role in Alzheimers disease (AD). It has been reported that autophagic flux is altered in patients with AD, and application of the autophagy enhancer rapamycin may alleviate the cognitive impairment and amyloid- (A) neuropathology in transgenic animal model of AD. Since rapamycin is also an immune suppressor, there is a concern that long-term use of rapamycin may bring severe unwanted side effects. The aim of this study is to test if carbamazepine (CBZ), an anti-epileptic drug that has a potent autophagy enhancement effect, has anti-AD effects in APPswe/PS1deltaE9 transgenic mice model of AD. We found that APPswe/PS1deltaE9 mice display increased autophagic activity accompanied by decreased mTOR activity. After three months treatment with CBZ in the APPswe/PS1deltaE9 mice, we demonstrated that the spatial learning and memory deficits in these mice are significantly alleviated. We also documented that the cerebral amyloid plaque burden and A42 levels in these mice are significantly reduced. Furthermore, we showed that CBZ significantly enhances the autophagic flux in the APPswe/PS1deltaE9 mice which is unlikely via mTOR-dependent autophagy pathway. These data suggest that long-term CBZ treatment may have a protective effect in AD mouse model possibly through enhancing the autophagic flux.

Human DNA polymerase δ (Pol δ) is involved in various DNA damage responses in addition to its central role in DNA replication. The Pol δ4 holoenzyme consists of four subunits, p125, p50, p68 and p12. It has been established that the p12 subunit is rapidly degraded in response to DNA damage by UV leading to the in vivo conversion of Pol δ4 to Pol δ3, a trimeric form lacking the p12 subunit. We provide the first analysis of the time-dependent recruitment of the individual Pol δ subunits to sites of DNA damage produced by UV irradiation through 5 μm polycarbonate filters by immunofluorescence microscopy and laser scanning cytometry (LSC). Quantitative analysis demonstrates that the recruitments of the three large subunits was near complete by 2 h and did not change significantly up to 4 h after UV exposure. However, the recruitment of p12 was incomplete even at 4 h, with about 70% of the Pol δ lacking the p12 subunit. ChIP analysis of Pol δ after global UV irradiation further demonstrates that only p125, p50 and p68 were present. Thus, Pol δ3 is the predominant form of Pol δ at sites of UV damage as a result of p12 degradation. Using LSC, we have further confirmed that Pol δ was recruited to CPD damage sites in all phases of the cell cycle. Collectively, our results show that Pol δ at the DNA damage site is the Pol δ trimer lacking p12 regardless of the cell cycle phase.

Ubiquitination of proliferating cell nuclear antigen (PCNA) to ub-PCNA is essential for DNA replication across bulky template lesions caused by UV radiation and alkylating agents, as ub-PCNA orchestrates the recruitment and switching of translesion synthesis (TLS) polymerases with replication polymerases. This allows replication to proceed, leaving the DNA to be repaired subsequently. Defects in a TLS polymerase, Pol η, lead to a form of Xeroderma pigmentosum, a disease characterized by severe skin sensitivity to sunlight damage and an increased incidence of skin cancer. Structurally, however, information on how ub-PCNA orchestrates the switching of these two classes of polymerases is lacking. We have solved the structure of ub-PCNA and demonstrate that the ubiquitin molecules in ub-PCNA are radially extended away from the PCNA without structural contact aside from the isopeptide bond linkage. This unique orientation provides an open platform for the recruitment of TLS polymerases through ubiquitin-interacting domains. However, the ubiquitin moieties, to the side of the equatorial PCNA plane, can place spatial constraints on the conformational flexibility of proteins bound to ub-PCNA. We show that ub-PCNA is impaired in its ability to support the coordinated actions of Fen1 and Pol δ in assays mimicking Okazaki fragment processing. This provides evidence for the novel concept that ub-PCNA may modulate additional DNA transactions other than TLS polymerase recruitment and switching.

The ubiquitination of PCNA is an essential event in the operation of the DNA Damage Tolerance (DDT) pathway that is activated after DNA damage caused by UV or chemical agents during S-phase. This pathway allows the bypass of DNA damage by translesion synthesis that would otherwise cause replication fork stalling. PCNA is mono-ubiquitinated by Rad18-Rad6, and polyubiquitinated by Rad5-Ubc13/Uev1 in the DDT pathway. Mono-and polyubiquitination of PCNA are key processes in the translesion bypass and template switching sub-pathways of the DDT. DNA damage by IR causes DSBs, which trigger the DNA Damage Response (DDR). The ubiquitin ligase RNF8 has a critical role in the assembly of BRCA1 complexes at the DSBs in the DDR. We show that RNF8 readily mono-ubiquitinates PCNA in the presence of UbcH5c, and polyubiquitinates PCNA in the added presence of Ubc13/Uev1a. These reactions are the same as those performed by Rad18-Rad6 and Rad5-Ubc13. RNF8 depletion suppressed both UV and MNNG-stimulated mono-ubiquitination of PCNA, revealing that an RNF8-dependent pathway for PCNA ubiquitination is operative in vivo. These findings provide evidence that RNF8, a key E3 ligase in the DDR, may also play a role in the DDT.

Abstract Experiments were carried out to study the morphological characters of Myzus persicae(Sulzer) on tobacco, rape and peach from different localities of China. The results showed that the number of accessory hairs on ultimate rostral segment had no difference among all samples in 14 morphological characters used by Blackman in this study. Cluster analysis indicated that the morphological differences between samples from peach and samples from other host plants was the biggest, the next was the samples from Changchun on tobacco. Compared with peach, the aphid from rape was more similar to that from tobacco in morphology. For the samples on tobacco from the central and southern China, they can be clustered into yellowish‐green group and reddish‐brown group. It shows unlikely to separate successfully the aphids on tobacco from those on non‐tobacco host plants only by morphological characters.

China has taken great strides over the last 15 years to constrain the rise of CO2emissions from its energy sector. Energy intensity has declined, the installed capacity for wind and solar energy has soared, and the share of fossil fuels is falling. This success has been achieved through deploying administrative policy instruments backed by generous state finance. As this approach is yielding diminishing returns, the government has launched a number of initiatives to increase the role of market forces across the energy sector: in the oil, gas and electricity industries, and through the development of carbon markets. However, the wider political and economic context is undermining these moves. Central and local government agencies regularly intervene to disrupt the operation of these markets. At the same time, most resources and assets remain in the hands of enterprises owned by the state at central or local levels, giving them too the ability to undermine these markets. The application of institutional concepts provides insights into how the style of governance at national level and within the energy sector constrains the development of the new market initiatives. As a result, these market mechanisms will not yield their potential efficiency improvements and emissions reductions in the current institutional context. These finding are also relevant to many other countries, especially those with a long history of state involvement in the energy sector.