E3HistoneLASU1, a 500 kDa novel multi-functional ubiquitin protein ligase

During spermatogenesis histones must be degraded in late round and early elongating spermatids to permit chromatin condensation. Ubiquitin conjugation is activated and histones are ubiquitinated at this stage, suggesting that histone degradation may be mediated by ubiquitination. The activation of ubiquitin conjugation during spermatogenesis is dependent on the ubiquitin conjugating enzyme (E2) UBC4. We therefore studied whether histones are ubiquitinated by a UBC4 dependent ubiquitin protein ligase (E3) during spermatogenesis. E3Histone was identified by a biochemical screen and purified to near homogeneity. Mass spectrometry identified E3Histone as LASU1, a 482 kDa HECT domain protein and E3Histone conjugates ubiquitin to all core histories in vitro. UBC4-1 and UBC4-testis were the preferred E2s for E3Histone-dependent ubiquitination of histones. E3Histone was the major UBC4-1 dependent histone ubiquitinating E3 in testis. Anti-LASU1 antibody immunodepleted E3 Histone activity. Immunohistochemistry showed that E3Histone /LASU1 was predominantly expressed in nuclei from spermatogonia to mid-pachytene cells, but not detectable in spermatids. Histones are also ubiquitinated in spermatocytes. E3Histone/LASU1 was widely expressed in different mouse tissues. It was mainly expressed in the cytoplasm in most tissues, except in neurons of the brain and in early germ cells of the testis where it was expressed in the nucleus. In most tissues, E3Histone/LASU1 was expressed in epithelia. The wide expression of E3Histone/LASU1 suggests the existence of substrates of this E3 other than histones. Indeed, our assays showed that in vitro purified E3Histone stimulates polyubiquitination of Mcl-1, a BH3 region containing antiapoptotic protein. E3Histone may therefore regulate cell apoptosis by mediating degradation of Mcl-1. Since E3Histone/LASU1 was found previously to affect gene transcription and histone monoubiquitination is known to regulate gene transcription, we also evaluated the role of E3 Histone/LASU1 in histone ubiquitination in somatic cells. Depletion of E3Histone/LASU1 protein by siRNA did not affect the levels of free or ubiquitinated histories. In summary, E3Histone /LASU1 is a novel multi-functional protein that may mediate histone ubiquitination during meiosis and may be involved in apoptosis by triggering Mcl-1 degradation. Its wide expression and large non-catalytic region indicate that there are likely many other substrates of E3Histone/LASU1

A Pathogen Secreted Protein as a Detection Marker for Citrus Huanglongbing.

The citrus industry is facing an unprecedented crisis due to Huanglongbing (HLB, aka citrus greening disease), a bacterial disease associated with the pathogen Candidatus Liberibacter asiaticus (CLas) that affects all commercial varieties. Transmitted by the Asian citrus psyllid (ACP), CLas colonizes citrus phloem, leading to reduced yield and fruit quality, and eventually tree decline and death. Since adequate curative measures are not available, a key step in HLB management is to restrict the spread of the disease by identifying infected trees and removing them in a timely manner. However, uneven distribution of CLas cells in infected trees and the long latency for disease symptom development makes sampling of trees for CLas detection challenging. Here, we report that a CLas secreted protein can be used as a biomarker for detecting HLB infected citrus. Proteins secreted from CLas cells can presumably move along the phloem, beyond the site of ACP inoculation and CLas colonized plant cells, thereby increasing the chance of detecting infected trees. We generated a polyclonal antibody that effectively binds to the secreted protein and developed serological assays that can successfully detect CLas infection. This work demonstrates that antibody-based diagnosis using a CLas secreted protein as the detection marker for infected trees offers a high-throughput and economic approach that complements the approved quantitative polymerase chain reaction-based methods to enhance HLB management programs

Integrating Strategies of Herbal Metabolomics, Network Pharmacology, and Experiment Validation to Investigate Frankincense Processing Effects

In-depth research on processing can promote the globalization of processed herbs. The purpose of this study is to propose an improved strategy for processing effect investigation. Frankincense and processed frankincense were used as research subjects. First, high-speed countercurrent chromatography (HSCCC) and preparation high-performance liquid chromatography (PHPLC) techniques were used for major compounds isolation and minor compounds concentration. Processed frankincense was subjected to two stepwise solvent systems, namely, n-hexane:ethanol:water (6:5:1) and n-hexane:methyl-acetate:acetonitrile:water (4:4:3:4), to yield 12 fractions, and 18 compounds were further separated. Second, a comprehensive metabolomic analysis conducted by ultrahigh-performance liquid-chromatography/electrospray-ionization mass spectrometry (UHPLC-Qtof-MS) coupled with multivariate statistics was performed to fully characterize the chemical components and discover the potential biomarkers between frankincense and processed frankincense. In total, 81 metabolites, including the 18 separated compounds, were selected as potential biomarkers between frankincense and processed frankincense among 153 detected compounds for their VIP values of greater than one. The tirucallane-type compounds and components with 9,11-dehydro structures clearly occurred at high levels in the processed frankincense, while lupine-type compounds and those with 11-keto structures were significantly higher in frankincense. Then, a network pharmacology model was constructed to decipher the potential mechanisms of processing. Intestinal absorption properties prediction indicated the possibility of processing-related absorption enhancement. A systematic analysis of the constructed networks showed that the C-T network was constructed with 18 potential biomarkers and 69 targets. TNF and IL-1β were among the top-ranked and were linked by 8 and 7 pathways, which were mainly involved in inflammation. The arachidonic acid metabolism pathway exhibited the highest number of target connections. Finally, the prediction was validated experimentally by an intestinal permeability and efficacy assay. The experiments provided convincing evidence that processed frankincense harbored stronger inhibition effects toward TNF-α-, IL-1β- and arachidonic acid-induced platelet aggregation. The processing procedure leads to changes of the chemical metabolites, which triggers the enhancement of absorption and cure efficiency. The global change of the metabolites, absorption and pharmacological effects of processing were depicted in a systematic manner

Gene co-expression network analysis identifies BRCC3 as a key regulator in osteogenic differentiation of osteoblasts through a β-catenin signaling dependent pathway

Objective(s): The prognosis of osteoporosis is very poor, and it is very important to identify a biomarker for prevention of osteoporosis. In this study, we aimed to identify candidate markers in osteoporosis and to investigate the role of candidate markers in osteogenic differentiation. Materials and Methods: Using Weighted Gene Co-Expression Network analysis, we identified three hub genes might associate with osteoporosis. The mRNA expression of hub genes in osteoblasts from osteoporosis patients or healthy donor was detected by qRT-PCR. Using siRNA and overexpression, we investigated the role of hub gene BRCC3 in osteogenic differentiation by alkaline phosphatase staining and Alizarin red staining. Moreover, the role of β-catenin signaling in the osteogenic differentiation was detected by using β-catenin signaling inhibitor XAV939.Results: We identified three hub genes that might associate with osteoporosis including BRCC3, UBE2N, and UBE2K. UBE2N mRNA and UBE2K mRNA were not changed in osteoblasts isolated from osteoporosis patients, compared with healthy donors, whereas BRCC3 mRNA was significantly increased. Depletion of BRCC3 promoted the activation of alkaline phosphatase and formation of calcified nodules in osteoblasts isolated from osteoporosis patients and up-regulated β-catenin expression. XAV939 reversed the BRCC3 siRNA-induced osteogenic differentiation. Additionally, inhibited osteogenic differentiation was also observed after BACC3 overexpression, and this was accompanied by decreased β-catenin expression.Conclusion: BRCC3 is an important regulator for osteogenic differentiation of osteoblasts through β-catenin signaling, and it might be a promising target for osteoporosis treatment

Effect of Chitosan Coating with Different Molecular Weights on the Storage Quality of Postharvest Passion Fruit (Passiflora edulis Sims)

To study the preservation effect of chitosan coating with different molecular weights on postharvest passion fruit, the "Qinmi No.9" was coated with chitosan of molecular weights of 30, 50, 100, 150 and 200 kDa (1.5%, w/v) to determine the quality of passion fruit during storage. The results showed that chitosan coating with different molecular weights was able to delay the shrinkage and yellowing, reduce the weight loss rate and inhibit the decay of passion fruit. Moreover, chitosan with a larger molecular weight was more conducive to delaying the ripening and senescence of passion fruit, as well as reducing shrinkage, and decay. At the end of storage, the weight loss of fruits coated with 200 kDa chitosan was nearly 10% less than that coated with 30 kDa chitosan, and the fruits coated with 150 and 200 kDa chitosan did not decay. The lower molecular weight (30 and 50 kDa) and higher molecular weight (150 kDa) chitosan were more effective in inhibiting weight loss, total soluble solids and soluble sugar metabolism, and maintaining titratable acid, flavonoid and total phenol contents of fruit during storage. The chitosan with 150 kDa had the best effect in maintaining the vitamin C content, which was 1.12 times higher than the control group at the end of storage. In conclusion, chitosan with different molecular weights was effective to delay senescence, slow down water loss and shrink of passion fruit and maintain the quality, chitosan with 150 kDa was more suitable to maintain the quality of postharvest passion fruit

Tumor Tissue-Derived Formaldehyde and Acidic Microenvironment Synergistically Induce Bone Cancer Pain

Background: There is current interest in understanding the molecular mechanisms of tumor-induced bone pain. Accumulated evidence shows that endogenous formaldehyde concentrations are elevated in the blood or urine of patients with breast, prostate or bladder cancer. These cancers are frequently associated with cancer pain especially after bone metastasis. It is well known that transient receptor potential vanilloid receptor 1 (TRPV1) participates in cancer pain. The present study aims to demonstrate that the tumor tissue-derived endogenous formaldehyde induces bone cancer pain via TRPV1 activation under tumor acidic environment. Methodology/Principal Findings: Endogenous formaldehyde concentration increased significantly in the cultured breast cancer cell lines in vitro, in the bone marrow of breast MRMT-1 bone cancer pain model in rats and in tissues from breast cancer and lung cancer patients in vivo. Low concentrations (1 similar to 5 mM) of formaldehyde induced pain responses in rat via TRPV1 and this pain response could be significantly enhanced by pH 6.0 (mimicking the acidic tumor microenvironment). Formaldehyde at low concentrations (1 mM to 100 mM) induced a concentration-dependent increase of [Ca(2+)]i in the freshly isolated rat dorsal root ganglion neurons and TRPV1-transfected CHO cells. Furthermore, electrophysiological experiments showed that low concentration formaldehyde-elicited TRPV1 currents could be significantly potentiated by low pH (6.0). TRPV1 antagonists and formaldehyde scavengers attenuated bone cancer pain responses. Conclusions/Significance: Our data suggest that cancer tissues directly secrete endogenous formaldehyde, and this formaldehyde at low concentration induces metastatic bone cancer pain through TRPV1 activation especially under tumor acidic environment.Multidisciplinary SciencesSCI(E)PubMed24ARTICLE4e10234

The impact of statin use before intensive care unit admission on patients with acute kidney injury after cardiac surgery

Background: Cardiac surgery-associated acute kidney injury (CSA-AKI) is a common and serious complication after cardiac surgery. The influence of statin use before surgery on the renal outcome of patients undergoing cardiac surgery is controversial. The purpose of this study was to evaluate the effect of statins on postoperative renal outcomes in patients undergoing cardiac surgery.Methods: We included CSA-AKI patients in the Medical Information Mart for Intensive Care (MIMIC)—IV database and were divided into statin group and non-statin group according to whether they used statins before entering intensive care units (ICU). The main outcomes were hospitalization and 30-day mortality, and the secondary outcomes were 60-day mortality and 90-day mortality. We used propensity score matching (PSM) to adjust for confounding factors. The 95% confidence interval (CI) and risk ratio (RO) were calculated by the COX proportional regression model. At the same time, stratified analysis was used to explore whether the relationship between the statins use before intensive care units and mortality was different in each subgroup and whether the relationship between different doses of Atorvastatin and mortality was different.Result: We identified 675 pre-ICU statin users and 2095 non-statin users. In the COX proportional regression model, pre-ICU statin use was associated with decreased in-hospital (HR = 0.407, 95%confidence interval 0.278–0.595, p < 0.001) and 30-day mortality (HR = 0.407, 95%CI 0.279–0.595, p < 0.001). The survival rate of patients who took statins before entering ICU was significantly higher than that of those who did not use statins at 30 days, 60 days and 90 days. There is a significant interaction between patients with aged>65 years (HR = 0.373, 95%CI 0.240–0.581, p < 0.001), Acute kidney injury grade I (HR = 0.244, 95%CI 0.118–0.428, p < 0.001), and without post-myocardial infarction syndrome (HR = 0.344, 95%CI 0.218–0.542, p < 0.001). The mortality in hospital and 60 days of CSA-AKI patients treated with ≥80 mg Atorvastatin before operation was significantly reduced (p < 0.05).Conclusion: The pre-ICU statin use was significantly associated with decreased risk in hospital and 30-day mortality. The preoperative use of ≥80 mg Atorvastatin may improve the prognosis of CSA-AKI

The molecular mechanism for inhibiting the growth of nasopharyngeal carcinoma cells using polymethoxyflavonoids purified from pericarp of Citrus reticulata ‘Chachi’ via HSCCC

Polymethoxyflavonoids (PMFs), the main bioactive compounds naturally occurring in the pericarp of Citrus reticulata ‘Chachi’ (CRCP), possess significant antitumor action. However, the action of PMFs in nasopharyngeal carcinoma (NPC) is currently unknown. The present research study was conducted to investigate the inhibitory mechanisms of PMFs from CRCP on NPC growth in vivo and in vitro. In our research, we used high-speed counter-current chromatography (HSCCC) to separate four PMFs (nobiletin (NOB), 3,5,6,7,8,3′,4′-heptamethoxyflavone (HMF), tangeretin (TGN), and 5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone (5-HPMF)) from CRCP. CCK-8 assay was used to preliminarily screen cell viability following exposure to the four PMFs. Colony formation, Hoechst-33258 staining, transwell, and wound scratch assays were performed to assess the anti-proliferation, invasion, migration, and apoptosis-inducing effects of HMF on NPC cells. NPC tumors in xenograft tumor transplantation experiments were also established to explore the effect of HMF (100 and 150 mg/kg/day) on NPC. The histopathological changes in the treated rats were observed by H&E staining and Ki-67 detection by immunohistochemical techniques. The expressions of P70S6K, p-P70S6K, S6, p-S6, COX-2, p53, and p-p53 were measured by Western blot. The four PMFs were obtained with high purity (>95.0%). The results of the preliminary screening by CCK-8 assay suggested that HMF had the strongest inhibitory effect on NPC cell growth. The results of the colony formation, Hoechst-33258 staining, transwell, and wound scratch assays indicated that HMF had significant anti-proliferation, invasion, migration, and apoptosis-inducing ability in NPC cells. Moreover, HMF suppressed NPC tumor growth in xenograft tumor transplantation experiments. Further investigation suggested that HMF regulated NPC cells proliferation, apoptosis, migration, and invasion by activating AMPK-dependent signaling pathways. In conclusion, HMF-induced AMPK activation inhibited NPC cell growth, invasion, and metastatic potency by downregulating the activation of the mTOR signaling pathway and COX-2 protein levels, as well as enhancing the p53 phosphorylation level. Our study provides a crucial experimental basis for the clinical treatment of NPC, as well as the development and utilization of PMFs from CRCP