Positive selection CRISPR screens reveal a druggable pocket in an oligosaccharyltransferase required for inflammatory signaling to NF-κB.

Nuclear factor κB (NF-κB) plays roles in various diseases. Many inflammatory signals, such as circulating lipopolysaccharides (LPSs), activate NF-κB via specific receptors. Using whole-genome CRISPR-Cas9 screens of LPS-treated cells that express an NF-κB-driven suicide gene, we discovered that the LPS receptor Toll-like receptor 4 (TLR4) is specifically dependent on the oligosaccharyltransferase complex OST-A for N-glycosylation and cell-surface localization. The tool compound NGI-1 inhibits OST complexes in vivo, but the underlying molecular mechanism remained unknown. We did a CRISPR base-editor screen for NGI-1-resistant variants of STT3A, the catalytic subunit of OST-A. These variants, in conjunction with cryoelectron microscopy studies, revealed that NGI-1 binds the catalytic site of STT3A, where it traps a molecule of the donor substrate dolichyl-PP-GlcNAc2-Man9-Glc3, suggesting an uncompetitive inhibition mechanism. Our results provide a rationale for and an initial step toward the development of STT3A-specific inhibitors and illustrate the power of contemporaneous base-editor and structural studies to define drug mechanism of action.

Toward a CRISPR-based mouse model of Vhl-deficient clear cell kidney cancer: Initial experience and lessons learned.

CRISPR is revolutionizing the ability to do somatic gene editing in mice for the purpose of creating new cancer models. Inactivation of the VHL tumor suppressor gene is the signature initiating event in the most common form of kidney cancer, clear cell renal cell carcinoma (ccRCC). Such tumors are usually driven by the excessive HIF2 activity that arises when the VHL gene product, pVHL, is defective. Given the pressing need for a robust immunocompetent mouse model of human ccRCC, we directly injected adenovirus-associated viruses (AAVs) encoding sgRNAs against VHL and other known/suspected ccRCC tumor suppressor genes into the kidneys of C57BL/6 mice under conditions where Cas9 was under the control of one of two different kidney-specific promoters (Cdh16 or Pax8) to induce kidney tumors. An AAV targeting Vhl, Pbrm1, Keap1, and Tsc1 reproducibly caused macroscopic ccRCCs that partially resembled human ccRCC tumors with respect to transcriptome and cell of origin and responded to a ccRCC standard-of-care agent, axitinib. Unfortunately, these tumors, like those produced by earlier genetically engineered mouse ccRCCs, are HIF2 independent.

HOXA9 gene inhibits proliferation and differentiation and promotes apoptosis of bovine preadipocytes.

BACKGROUND: Hox gene family is an important transcription factor that regulates cell process, and plays a role in the process of adipocytes differentiation and fat deposition. Previous transcriptome sequencing studies have indicated that the Homeobox A9 gene (HOXA9) is a candidate gene for regulating the process of bovine lipid metabolism, but the function and specific mechanism of action remain unclear. Therefore, this study aims to explore the role of HOXA9 in the proliferation, differentiation and apoptosis of bovine preadipocytes through gain-of-function and lose-of-function. RESULT: It found HOXA9 highly expressed in bovine adipose tissue, and its expression level changed significantly during adipocytes differentiation process. It gave a hint that HOXA9 may be involved in the process of bovine lipid metabolism. The results of HOXA9 gain-of-function experiments indicated that HOXA9 appeared to act as a negative regulator not only in the differentiation but also in the proliferation of bovine preadipocytes, which is mainly reflected that overexpression of HOXA9 down-regulate the mRNA and protein expression level of PPARγ, CEBPα and FABP4 (P < 0.05). The mRNA expression level of CDK1, CDK2, PCNA, CCNA2, CCNB1, CCND1 and CCNE2, as well as the protein expression of CDK2 also significantly decreased. The decrease of lipid droplets content was the main characteristic of the phenotype (P < 0.01), which further supported the evidence that HOXA9 was a negative regulator of preadipocytes differentiation. The decrease of cell proliferation rate and EdU positive rate, as well as the limitation of transition of preadipocytes from G0/G1 phase to S phase also provided evidence for the inhibition of proliferation. Apart from this above, we noted an interesting phenomenon that overexpression of HOXA9 showed in a significant upregulation of both mRNA and protein level of apoptosis markers, accompanied by a significant increase in cell apoptosis rate. These data led us not to refute the fact that HOXA9 played an active regulatory role in apoptosis. HOXA9 loss-of-function experiments, however, yielded the opposite results. Considering that HOXA9 acts as a transcription factor, we predicted its target genes. Dual luciferase reporter assay system indicated that overexpression of HOXA9 inhibits activity of PCNA promoter. CONCLUSION: Taken together, we demonstrated for the first time that HOXA9 played a role as a negative regulatory factor in the differentiation and proliferation of preadipocytes, but played a positive regulatory role in apoptosis, and it may play a regulatory role by targeting PCNA. This study provides basic data for further exploring the regulatory network of intramuscular fat deposition in bovine.

Integrated transcriptome and DNA methylome analysis reveal the biological base of increased resistance to gray leaf spot and growth inhibition in interspecific grafted tomato scions.

BACKGROUND: Grafting is widely used as an important agronomic approach to deal with environmental stresses. However, the molecular mechanism of grafted tomato scions in response to biotic stress and growth regulation has yet to be fully understood. RESULTS: This study investigated the resistance and growth performance of tomato scions grafted onto various rootstocks. A scion from a gray leaf spot-susceptible tomato cultivar was grafted onto tomato, eggplant, and pepper rootstocks, creating three grafting combinations: one self-grafting of tomato/tomato (TT), and two interspecific graftings, namely tomato/eggplant (TE) and tomato/pepper (TP). The study utilized transcriptome and DNA methylome analyses to explore the regulatory mechanisms behind the resistance and growth traits in the interspecific graftings. Results indicated that interspecific grafting significantly enhanced resistance to gray leaf spot and improved fruit quality, though fruit yield was decreased compared to self-grafting. Transcriptome analysis demonstrated that, compared to self-grafting, interspecific graftings triggered stronger wounding response and endogenous immune pathways, while restricting genes related to cell cycle pathways, especially in the TP grafting. Methylome data revealed that the TP grafting had more hypermethylated regions at CHG (H = A, C, or T) and CHH sites than the TT grafting. Furthermore, the TP grafting exhibited increased methylation levels in cell cycle related genes, such as DNA primase and ligase, while several genes related to defense kinases showed decreased methylation levels. Notably, several kinase transcripts were also confirmed among the rootstock-specific mobile transcripts. CONCLUSIONS: The study concludes that interspecific grafting alters gene methylation patterns, thereby activating defense responses and inhibiting the cell cycle in tomato scions. This mechanism is crucial in enhancing resistance to gray leaf spot and reducing growth in grafted tomato scions. These findings offer new insights into the genetic and epigenetic contributions to agronomic trait improvements through interspecific grafting.

Genome-wide identification of oxidosqualene cyclase genes regulating natural rubber in Taraxacum kok-saghyz.

MAIN CONCLUSION: Nine TkOSC genes have been identified by genome-wide screening. Among them, TkOSC4-6 might be more crucial for natural rubber biosynthesis in Taraxacum kok-saghyz roots. Taraxacum kok-saghyz Rodin (TKS) roots contain large amounts of natural rubber, inulin, and valuable metabolites. Oxidosqualene cyclase (OSC) is a key member for regulating natural rubber biosynthesis (NRB) via the triterpenoid biosynthesis pathway. To explore the functions of OSC on natural rubber producing in TKS, its gene family members were identified in TKS genome via genome-wide screening. Nine TkOSCs were identified, which were mainly distributed in the cytoplasm. Their family genes experienced a neutral selection during the evolution process. Overall sequence homology analysis OSC proteins revealed 80.23% similarity, indicating a highly degree of conservation. Pairwise comparisons showed a multiple sequence similarity ranging from 57% to 100%. Protein interaction prediction revealed that TkOSCs may interact with baruol synthase, sterol 1,4-demethylase, lupeol synthase and squalene epoxidase. Phylogenetic analysis showed that OSC family proteins belong to two branches. TkOSC promoter regions contain cis-acting elements related to plant growth, stress response, hormones response and light response. Protein accumulation analysis demonstrated that TkOSC4, TkOSC5 and TkOSC6 proteins had strong expression levels in the root, latex and plumular axis. Comparison of gene expression patterns showed TkOSC1, TkOSC4, TkOSC5, TkOSC6, TkOSC7, TkOSC8 and TkOSC9 might be important in regulating NRB. Combination of gene and protein results revealed TkOSC4-6 might be more crucial, and the data might contribute to a more profound understanding of the roles of OSCs for NRB in TKS roots.

A Rotating Triboelectric Nanogenerator Driven by Bidirectional Swing for Water Wave Energy Harvesting.

Due to the simple installation and convenient maintenance, the floating water wave energy harvesting devices have significant economic advantages. Mass power density is the most important index to evaluate the advancement of floating wave energy harvesting devices. Herein, a self-adaptive rotating triboelectric nanogenerator (SR-TENG) with a compound pendulum and a functional gear-set is provided for wave energy harvesting. First, a compound pendulum structure with a low center of gravity and high moment of inertia is obtained by the geometric design and mechanical study. Besides, compared with the previous triboelectric nanogenerator with one-way clutch, SR-TENG can harvest twice the kinetic energy utilization through the functional gear-set. Importantly, depending on the structure design, the SR-TENG obtains the average mass power density of 45.18 mW kg-1 under low frequency driving conditions, which is about 10 times the reference electromagnetic generator with a similar structure and size. This result shows that the SR-TENG has a significant advantage in small water wave energy harvesting. These findings provide an important example of the floating water wave energy harvesting devices.

Predictor of cognitive impairment: metabolic syndrome or circadian syndrome.

BACKGROUND: It was reported that metabolic syndrome increases the risk for cognitive impairment and circadian rhythm may influence cognition behavior. Identifying the potential risk factors is essential to screen individuals with neuronal dysfunction, neuronal loss, and cognitive decline and prevent cognitive impairment and dementia development. METHODS: We clarified participants by the presence of metabolic syndrome (MetS) and circadian syndrome (CircS) and employed three multivariable Generalized Estimating Equation (GEE) models to control the potential confounding factors and estimate the ß values for cognitive function using as referents those had neither MetS nor CircS at baseline. The cognitive function consists of episodic memory and executive function was estimated via the modified Telephone Interview for Cognitive Status (TICS) every two years until 2015. RESULTS: The mean age of the participants was 58.80 (8.93) years and 49.92% (male). The prevalence of MetS and CircS was 42.98% and 36.43%, respectively. 1,075 (11.00%) and 435 (4.45%) participants had either MetS or CircS alone and 3,124 (31.98%) had both CircS and MetS. Participants with both MetS and CircS compared with normal had a significantly decreased cognitive function score during the 4-years cohort (ß = -0.32, 95% CI: -0.63, -0.01) with the complete model, as well as among participants who suffered from CircS alone (ß = -0.82, 95% CI: -1.47, -0.16), while not among participants with MetS alone (ß = 0.13, 95% CI: -0.27, 0.53). Specifically, compared with the normal population a significantly lower score was discovered in the episodic memory (ß = -0.51, 95% CI: -0.95, -0.07), while slightly lower in executive function (ß = -0.33, 95% CI: -0.68, -0.01) among individuals with CircS alone. CONCLUSIONS: Individuals with CircS alone or both MetS and CircS have a high risk of cognitive impairment. The association was even stronger in participants with CircS alone than those with both MetS and CircS, suggesting CircS probably have a stronger association with cognitive functioning than MetS and could be a better predictor for cognitive impairment.

Safe Rice Production in Cd-Contaminated Paddy Soil: Strategy and Environmental Implications.

This field study explored safe rice production in Cd-contaminated red paddy soil by application of the combined Si-/Se- containing foliar inhibitors (Si or Se) and the mixture amendments of quicklime (Q), polyacrylamide (A), or/and sepiolite (S) at low (1) and high (2) application rates. The results showed that all treatments increased soil pH and decreased total P and soil organic matter (excluding QSe2). With the increasing application rates, QAS significantly decreased the available Cd because of the enhanced stabilization, while QSi and QSe significantly increased the available Cd because of the inhibited plant uptake. After remediation, QA1, QSi2, and QSe2 most effectively decreased the uptake Cd by rice to meet the threshold of National Food Safety Standard of China. The treatments excluding Q1, QA1, QSi1, and QSi2 did not dramatically change the bacterial community structure in soil. Collectively, QSe2 was recommended for remediating Cd-contaminated red paddy soil.

Preparation of a Large Amount of Ultrathin Graphdiyne.

We report a facile strategy to prepare a large amount of ultrathin graphdiyne (GDY) with good crystallinity in the mixture of oil-water systems. By simply mixing the solution of GDY monomer in CHCl3 and the copper acetate solution as a catalyst in water and stirring at room temperature, ultrathin GDY films with a thickness of ∼4 nm were obtained in a yield of 95 %. This work provides a feasible path for the substantial preparation of GDY films and may pave the way to the development of substantial preparation of the GDY materials.

BolTLP1, a Thaumatin-like Protein Gene, Confers Tolerance to Salt and Drought Stresses in Broccoli (Brassica oleracea L. var. Italica).

Plant thaumatin-like proteins (TLPs) play pleiotropic roles in defending against biotic and abiotic stresses. However, the functions of TLPs in broccoli, which is one of the major vegetables among the B. oleracea varieties, remain largely unknown. In the present study, bolTLP1 was identified in broccoli, and displayed remarkably inducible expression patterns by abiotic stress. The ectopic overexpression of bolTLP1 conferred increased tolerance to high salt and drought conditions in Arabidopsis. Similarly, bolTLP1-overexpressing broccoli transgenic lines significantly improved tolerance to salt and drought stresses. These results demonstrated that bolTLP1 positively regulates drought and salt tolerance. Transcriptome data displayed that bolTLP1 may function by regulating phytohormone (ABA, ethylene and auxin)-mediated signaling pathways, hydrolase and oxidoreductase activity, sulfur compound synthesis, and the differential expression of histone variants. Further studies confirmed that RESPONSE TO DESICCATION 2 (RD2), RESPONSIVE TO DEHYDRATION 22 (RD22), VASCULAR PLANT ONE-ZINC FINGER 2 (VOZ2), SM-LIKE 1B (LSM1B) and MALATE DEHYDROGENASE (MDH) physically interacted with bolTLP1, which implied that bolTLP1 could directly interact with these proteins to confer abiotic stress tolerance in broccoli. These findings provide new insights into the function and regulation of bolTLP1, and suggest potential applications for bolTLP1 in breeding broccoli and other crops with increased tolerance to salt and drought stresses.

Associations of C-reactive protein and fibrinogen with mortality from all-causes, cardiovascular disease and cancer among U.S. adults.

C-reactive protein (CRP) and fibrinogen are associated with an increased risk of death with suggested differences by gender, diet quality and race/ethnicity. However, the current evidence is limited. We used data including 8646 men and 9880 women from the National Health and Nutrition Examination Survey (NHANES) Linked Morality cohort (1999-2011) to investigate the associations of CRP and fibrinogen with mortality overall and by gender, race/ethnicity and diet quality. Cox-proportional hazard model was used to quantify the associations. With a median follow-up of 6 years, a strong dose-response relationship was observed between CRP levels and mortality risk in men after multivariable adjustment. For subjects who survived the first two years, the adjusted hazard ratios (HRs) for total mortality across quartiles (from lower to higher) of CRP were 1.97 (95% CI: 0.62-6.33), 1.89 (0.59, 6.06) and 6.34 (2.28-17.7) (P for trend <0.001). For cardiovascular disease (CVD) mortality, its association with CRP varied by diet quality. For cancer mortality, its association differed by history of cancer, and positive associations were observed only among subjects with history of cancer. In contrast, no such association of CRP with mortality was found in women. For fibrinogen, we observed its positive association with total mortality and the HRs across quartiles of fibrinogen (from lower to higher) were 1.21 (0.88, 1.67), 1.49 (1.22, 1.82) and 1.99 (1.56, 2.55). The association with CVD mortality differed by diet quality whereas no association was found with cancer mortality. Our findings suggest that higher levels of CRP and fibrinogen were associated with lower survival from total and CVD; the associations of CRP with mortality were more pronounced in men than women. Diet quality is an effect modifier for the association of CRP and fibrinogen with CVD mortality.

Distribution of high-risk HPV types among women in Sichuan province, China: a cross-sectional study.

BACKGROUND: Persistent infection with high-risk human papillomavirus (HR-HPV) is a major cause of cervical intraepithelial neoplasia and invasive cervical cancer. We investigated the prevalence of HR-HPV infection and distribution of viral genotypes among women in this area. METHODS: Women in Sichuan older than 20 years were screened for cervical cancer between January 2015 and December 2016 using liquid-based cytology testing and a flow cytometry-fluorescence hybridization test for HPV-DNA. Frequency tables were evaluated using the chi-squared test (χ2). RESULTS: Of the 17,319 women aged 20-85 years who participated in the study, Overall prevalence of HR-HPV infection was 12.6% (2178/17,319). The most prevalent viral type was HPV-52, which was present in 2.5% of individuals, followed by HPV-53 (1.6%), HPV-58 (1.5%), HPV-16 (1.1%), HPV-56 (0.9%), HPV-39 (0.8%). In HSIL, the five most common HR-HPV types were HPV52, 16, 58, 33 and 56. HPV16/18 in HSIL only makes up 25.9% of HSIL, whereas HPV31/33/45/52/58 make up 56.8%. Overall HR-HPV prevalence among women older than 65 years was 15.2%, significantly higher than the prevalence in other age groups (P < 0.05). Infection with dual or multiple HR-HPV types was associated with greater risk of abnormal cytology. CONCLUSION: Overall HR-HPV prevalence in Sichuan is as high as in large cities in China. The HR-HPV types 52, 16, 58, 33 and 56 predominated in this sample of HSIL women primarily from the banking and public sector in Sichuan. High prevalence among women older than 65 years needed pay attention to.

Radioprotective Effect of Walnut Oligopeptides Against Gamma Radiation-Induced Splenocyte Apoptosis and Intestinal Injury in Mice.

Walnut oligopeptides (WOPs) intake is associated with the augment of the antioxidant defense system and immune system. The chief object of this study is to evaluate the radioprotective effect of walnut oligopeptides extracted from walnut seed protein against 60Coγ-irradiation induced damage in mice. Female BALB/c mice were administered WOPs through drinking water for 14 days until a single dose of whole-body 60Coγ-irradiation. The 30-day survival test was carried out in the first group (8 Gy), and the other two groups (3.5 Gy) were sacrificed at 3 days and 14 days post-irradiation. Blood and organ samples of mice in the three groups were collected, the histopathological analysis and immunohistochemistry were conducted. The number of peripheral blood leukocytes, bone marrow DNA content, inflammatory cytokines, antioxidant capacity, and intestinal permeability were measured. We found that the administration of WOPs augmented antioxidant defense system, accelerated hematopoietic recovery and showed the significant trend toward higher survival rate and less weight loss compared with non-administrated control mice. In addition, WOPs administration appeared to be important to limit IR-induced splenocyte apoptosis and inflammatory cascade as well as reduce intestine epithelial barrier dysfunction and promote epithelial integrity. These results suggest that pre and post-treatment of WOPs may help to ameliorate acute damage, which is induced by ionizing radiation in mice and accelerate its recovery.

Knowledge of HPV and acceptability of HPV vaccine among women in western China: a cross-sectional survey.

BACKGROUND: Since most cervical cancer cases are caused by persistent high-risk human papillomavirus (HR-HPV) infection, knowledge of HPV among women is essential for the prevent of cervical cancer. This study was aimed to assess knowledge among women in western China about HPV and its association with cervical cancer, and to assess their acceptance of HPV vaccination. METHODS: A sample of healthy women undergoing routine physical examinations in the Health Management Center of West China Hospital, Sichuan University between January and December 2014 completed a questionnaire. RESULTS: A total of 1300 questionnaires were distributed, and 1109 were completed and analyzed. Only 28.85% of respondents (n = 320) had heard of HPV; among this subgroup, only half (53.44%) knew that it causes cervical cancer, only 26 (8.13%) correctly answered all questions about HPV. Multivariate analysis showed that respondents who had heard of HPV were more likely than other respondents to have a family history of any cancer, to undergo regular Pap tests and to have completed at least secondary education. Half of all respondents (51.22%) reported that they would be willing to be vaccinated against HPV. CONCLUSION: Although most women in western China lack basic knowledge about HPV, at least half are willing to take the HPV vaccine. Public health efforts to educate the public about HPV and its connection to cervical cancer should be strengthened and expanded.

Topology-defined units in numerosity perception.

What is a number? The number sense hypothesis suggests that numerosity is "a primary visual property" like color, contrast, or orientation. However, exactly what attribute of a stimulus is the primary visual property and determines numbers in the number sense? To verify the invariant nature of numerosity perception, we manipulated the numbers of items connected/enclosed in arbitrary and irregular forms while controlling for low-level features (e.g., orientation, color, and size). Subjects performed discrimination, estimation, and equality judgment tasks in a wide range of presentation durations and across small and large numbers. Results consistently show that connecting/enclosing items led to robust numerosity underestimation, with the extent of underestimation increasing monotonically with the number of connected/enclosed items. In contrast, grouping based on color similarity had no effect on numerosity judgment. We propose that numbers or the primitive units counted in numerosity perception are influenced by topological invariants, such as connectivity and the inside/outside relationship. Beyond the behavioral measures, neural tuning curves to numerosity in the intraparietal sulcus were obtained using functional MRI adaptation, and the tuning curves showed that numbers represented in the intraparietal sulcus were strongly influenced by topology.

What determines the object-level visual masking: The bottom-up role of topological change.

Object substitution masking (OSM) is said to occur on an object level without a close spatiotemporal proximity of target and mask. An influential account for OSM is "object updating," which espouses that OSM occurs when the target is updated by the mask as they share a single object representation. However, it is unclear what attribute determines whether the mask shares the same object representation as the target. We hypothesize that topological property determines whether a new object representation is built, and hence topological perception modulates object-level masking. We systematically manipulated the similarity between the target and the mask by changing a topological property (number of holes), color, shape, and orientation. We found that the topological change between the target and the mask reduced masking effects of all the other properties. Changing color, shape, or orientation, however, did not affect the masking effect of any other property. The global effect of the topological change remained across a variety of temporal and spatial distances between the target and the mask and was not limited to masking paradigms. Thus, our results suggest that the object representation, constrained by its topological properties, serves as a higher and global level of OSM, influencing the ongoing visual processing of features that are at a lower and local level.

Comparative Transcriptome Analysis Reveals an Efficient Mechanism of α-Linolenic Acid in Tree Peony Seeds.

Tree peony (Paeonia section Moutan DC.) species are woody oil crops with high unsaturated fatty acid content, including α-linolenic acid (ALA/18:3; >40% of the total fatty acid). Comparative transcriptome analyses were carried out to uncover the underlying mechanisms responsible for high and low ALA content in the developing seeds of P. rockii and P. lutea, respectively. Expression analysis of acyl lipid metabolism genes revealed upregulation of select genes involved in plastidial fatty acid synthesis, acyl editing, desaturation, and triacylglycerol assembly in seeds of P. rockii relative to P. lutea. Also, in association with ALA content in seeds, transcript levels for fatty acid desaturases (SAD, FAD2, and FAD3), which encode enzymes necessary for polyunsaturated fatty acid synthesis, were higher in P. rockii compared to P. lutea. Furthermore, the overexpression of PrFAD2 and PrFAD3 in Arabidopsis increased linoleic and ALA content, respectively, and modulated the final ratio 18:2/18:3 in the seed oil. In conclusion, we identified the key steps and validated the necessary desaturases that contribute to efficient ALA synthesis in a woody oil crop. Together, these results will aid to increase essential fatty acid content in seeds of tree peonies and other crops of agronomic interest.

Biochemical and Comparative Transcriptomic Analyses Identify Candidate Genes Related to Variegation Formation in Paeonia rockii.

Paeonia rockii is a wild tree peony species with large and dark purple variegations at the base of its petals. It is the genetic resource for various variegation patterns in tree peony cultivars, which is in contrast to the pure white petals of Paeonia ostii. However, the molecular mechanism underlying the formation of variegation in this plant is still unknown. Here, we conducted Illumina transcriptome sequencing for P. rockii, P. ostii (with pure white petals) and their F1 individuals (with purple-red variegation). A total of 181,866 unigenes were generated, including a variety of unigenes involved in anthocyanin biosynthesis and sequestration and the regulation of anthocyanin biosynthesis. The dark purple or purple-red variegation patterns mainly occurred due to the proportions of cyanidin (Cy)- and peonidin (Pn)-based anthocyanins. The variegations of P. rockii exhibited a "Cy > Pn" phenotype, whereas the F1 progeny showed a "Pn > Cy" phenotype. The CHS, DFR, ANS, and GST genes might play key roles in variegation pigmentation in P. rockii according to gene expression and interaction network analysis. Two R2R3-MYB transcription factors (c131300.graph_c0 and c133735.graph_c0) regulated variegation formation by controlling CHS, ANS and GST genes. Our results indicated that the various variegation patterns were caused by transcriptional regulation of anthocyanin biosynthesis genes, and the transcription profiles of the R2R3-MYBs provided clues to elucidate the mechanisms underlying this trait. The petal transcriptome data produced in this study will provide a valuable resource for future association investigations of the genetic regulation of various variegation patterns in tree peonies.

[Expression of miR-429 and Its Target Gene HSPA4L in Sperms from Asthenospermia Patients.]

OBJECTIVES: To investigate the expression of miR-429 and its target gene heat shock protein A4L (HSPA4L) in sperms from asthenospermia patients. METHODS: Twenty semen samples from healthy and fertile adults and 20 semen samples from asthenospermia patients were collected,and normal sperm parameters were defined according to World Health Organization criteria.The expression levels of miR-429 and HSPA4L mRNA were determined by qRT-PCR,and the bioinformatics tool (Targetscan) was used to predict the target of miR-429.Luciferase reporter assay and transfection study were performed to confirm target gene of miR-429.The expression levels of HSPA4L mRNA and protein were further determined by qRT-CPR and Western blot,respectively. RESULTS: The motility and viability of sperms from asthenospermia patients were lower than that in control group,and miR-429 was up-regulated in sperms from asthenospermia patients.Bioinformatics analysis revealed that HSPA4L was a target of miR-429.Luciferase reporter assay and transfection study further confirmed that miR-429 suppresses the expressions of HSPA4L mRNA and protein via directly targeting HSPA4L 3'UTR.Results from clinical samples also demonstrated that HSPA4L mRNA and protein were down-regulated in sperms from asthenospermia patients and the expression level of miR-429 was inversely correlated with the expression level of HSPA4L mRNA (r=-0.725, P<0.05). CONCLUSIONS: miR-429 is up-regulated in sperms from asthenospermia patients,and it may modulate the motility and viability of sperms via suppressing the expression of HSPA4L.

Residual amount of HBV DNA in serum is related to relapse in chronic hepatitis B patients after cessation of nucleos(t)ide analogs.

OBJECTIVE: To investigate the relationship between relapse and the levels of the residual amount of HBV DNA in serum at cessation in chronic hepatitis B patients meeting 2008 Asian Pacific Association for the Study of the Liver (APASL) nucleos(t)ide analogs (NAs) cessation criteria. METHODS: A total of 72 chronic hepatitis B patients who took NAs and had reached 2008 APASL cessation criteria entered the study. Patients were followed up for 6 months or longer after antiviral therapy was stopped. Serum HBV DNA level at cessation was detected by a highly sensitive polymerase chain reaction assay with detection limitation of 2 IU/mL. RESULTS: Of all the 72 patients, 42 patients (65.3%) relapsed after NA cessation. The detectable rate of the trace amount of HBV DNA at cessation was 41.7% by highly sensitive polymerase chain reaction reagents. The detectable rate of patients with consolidation treatment duration of <18 months was higher than that with consolidation duration of ≥18 months (47.5% vs. 15.4%, P=0.034), and the detectable rate of patients with HBeAg seroconversion within 6 months of treatment was lower than that of ≥6 months (25.0% vs. 61.5%, P=0.036). The residual amount of HBV DNA and detectable rate at cessation showed significant differences between relapsed and nonrelapsed patients (130.4±420.90 vs 44.6±155.16 IU/mL, P=0.004; 55.3% vs. 16.0%, P=0.001). The cutoff value predicting relapse was 2.24 IU/mL, with a sensitivity of 0.553 and specificity of 0.840. CONCLUSIONS: Residual amount of HBV DNA in serum at NA cessation is associated with HBV relapse. The cutoff value predicting relapse was 2.24 IU/mL, with a sensitivity of 0.553 and specificity of 0.840.