Aquaporin 1 is renoprotective in septic acute kidney injury by attenuating inflammation, apoptosis and fibrosis through inhibition of P53 expression.

Sepsis associated Acute kidney injury (AKI) is a common clinical syndrome characterized by suddenly decreased in renal function and urinary volume. This study was designed to investigate the role of Aquaporin 1 (AQP1) and P53 in the development of sepsis-induced AKI and their potential regulatory mechanisms. Firstly, transcriptome sequencing analysis of mice kidney showed AQP1 expression was reduced and P53 expression was elevated in Cecal ligation and puncture (CLP)-induced AKI compared with controls. Bioinformatics confirmed that AQP1 expression was remarkably decreased and P53 expression was obviously elevated in renal tissues or peripheral blood of septic AKI patients. Moreover, we found in vivo experiments that AQP1 mRNA levels were dramatically decreased and P53 mRNA significantly increased following the increased expression of inflammation, apoptosis, fibrosis, NGAL and KIM-1 at various periods in septic AKI. Meanwhile, AQP1 and P53 protein levels increased significantly first and then decreased gradually in kidney tissue and serum of rats in different stages of septic AKI. Most importantly, in vivo and vitro experiments demonstrated that silencing of AQP1 greatly exacerbates renal or cellular injury by up-regulating P53 expression promoting inflammatory response, apoptosis and fibrosis. Overexpression of AQP1 prevented the elevation of inflammation, apoptosis and fibrosis by down-regulating P53 expression in Lipopolysaccharide (LPS)-induced AKI or HK-2 cells. Therefore, our results suggested that AQP1 plays a protective role in modulating AKI and can attenuate inflammatory response, apoptosis and fibrosis via downregulating P53 in septic AKI or LPS-induced HK-2cells. The pharmacological targeting of AQP1 mediated P53 expression might be identified as potential targets for the early treatment of septic AKI.

New strategies and methods for reconstruction of the outer ear canal (OEC).

Background: The incidence of re-stenosis or re-atresia after reconstruction of the Outer Ear Canal (OEC) in patients with Congenital Malformation of the Middle and Outer Ear (CMMOE) is very high (up to 48%), and it has been a difficult problem for otologists not being able to solve.Aims/Objectives: To explore new strategies and methods to improve re-stenosis or re-atresia after reconstruction of the OEC in patients with CMMOE.Material and Methods: According to the characteristics of reconstructed OEC (r-OEC) re-stenosis or re-atresia summarized by us, a number of new prevention strategies and methods have been proposed and related patent products have been designed, including the improvement of covering epithelium types and skin grafting methods (7 types), simulated drum ring function to prevent the formation of negative pressure in the cavity, and strengthen postoperative support to reduce skin shrinkage and bone hyperplasia. The postoperative effects of different ages and preoperative OEC malformations are statistically analyzed.Results: The incidence of re-stenosis/re-atresia is 14.3% (5/35) in the thin sectional skin of the temporal scalp overlap splicing skin grafting, which was significantly better than 45.5% (15/33) in the whole piece mosaic splicing and barrel skin grafting from the inner thin sectional thigh skin and overlay splicing other methods, including the inner thigh thin sectional skin, chest medium thick skin and subcutaneous pedicle + chest medium thick skin (p<0.05). The patent artificial drum ring and the model stent of the OEC have obvious effects. The mean operation age of postoperative atresia, stenosis, and good groups are 9.3, 13.1, and 12.5 years old, respectively. The proportion of preoperative atresia is 91.3%, 85.7%, and 57.7%, respectively. The total incidence of re-atresia and re-stenosis of r-OEC for two groups of atresia and stenosis of OEC before surgery is 40.5% (49/121) and 13.3% (8/60), respectively.Conclusions and Significance: The best result is found in overlapping the splicing thin sectional skin of the temporal scalp, combined with artificial drum ring implantation, effective support of postoperative model stent of OEC and post-pubertal surgery selection are new and effective strategies and methods to prevent re-stenosis or re-atresia of r-OEC. Atresia or stenosis of the OEC before the operation is the influence factor of the postoperative effect.

[Effects of mouth opening breathing for different reasons on maxillofacial development in children].

Objective:To explore the effects of mouth opening breathing for different reasons on children's maxillofacial development. Methods:One hundred and fifty-one children were selected as the research objects of this experiment. They were divided into 49 cases of adenoid hypertrophy group（group A）, 52 cases of tonsillar hypertrophy group（group B） and 50 cases of adenoid with tonsillar hypertrophy group（Group C）. Healthy children in the same period were selected as the control group, a total of 45 cases. The reflex nasopharyngeal measurement parameters, facial development indexes and cephalometric parameters of group A, group B, group C and control group were analyzed, and the incidence of Angle ClassⅡand Angle Class Ⅲ in group A, group B and group C were studied. Results:Compared with the control group, the reflex nasopharyngeal measurement parameters in group A, group B and group C was significantly different（P<0.05）, and the cephalometric parameters changed with variation in groups（P<0.05）. The incidence of Angle Class Ⅱ facial pattern in group A and group C was higher, but the incidence of Angle Class Ⅲ facial pattern in group B and group C was higher（P<0.05）. Conclusion:Adenoid hypertrophy leads to mandibular retraction; tonsil hypertrophy leads to anterior mandibular arch; adenoid hypertrophy and tonsil hypertrophy are easy to lead to clockwise rotation of the mandible. In clinical practice, to avoid children's uncoordinated maxillofacial development, we should correct the maxillofacial situation of children as soon as possible.

Endotoxin induced acute kidney injury modulates expression of AQP1, P53 and P21 in rat kidney, heart, lung and small intestine.

This study was designed to explore whether aquaporin 1(AQP1), P53 and P21 can be used as diagnostic biomarkers of lipopolysaccharide (LPS)-induced acute kidney injury (AKI) and potential indicators of sepsis-induced multiple organ injury. Bioinformatics results demonstrated that AQP1, P53, P21 was dramatically elevated 6h after Cecal ligation and puncture (CLP)-AKI in rat renal tissue. The expression of AQP1, P53, P21, NGAL and KIM-1 in kidney were increased significantly at first and then decreased gradually in LPS-induced AKI rats. Histopathological sections showed swelling of tubular epithelial cells and destruction of basic structures as well as infiltration of numerous inflammatory cells in LPS-induced AKI. Moreover, the expressions of AQP1, P53 and P21 in heart were significantly increased in LPS treatment rats, while the AQP1 expressions in lung and small intestine were significantly decreased. The level of NGAL mRNA in heart, lung and small intestine was firstly increased and then decreased during LPS treatment rats, but the expression of KIM-1 mRNA was not affected. Therefore, our results suggest that AQP1, P53 and P21 is remarkably upregulated in LPS-induced AKI, which may be considered as a potential novel diagnostic biomarker of Septic AKI. NGAL may serve as a biomarker of sepsis-induced multiple organ damage during the process of LPS-induced AKI.

Aquaporin 1 Facilitates Ferroptosis, M1 Polarization, Mitochondrial Dysfunction, and Autophagy Damage on Lipopolysaccharide-Induced Macrophage Through Down-Regulation of P53 Signaling Pathway.

This study was designed to investigate the role of aquaporin 1 (AQP1) in ferroptosis, macrophage polarization, mitochondrial dysfunction, and impaired autophagy of lipopolysaccharide (LPS)-stimulated RAW264.7 cells and explored the underlying mechanisms. Si-AQP1-mediated AQP1 silencing RAW264.7 cells was constructed. Si-P53-mediated P53 silencing or pcDNA-P53 overexpression RAW264.7 cells was constructed. Assays of ATP, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and Mitochondrial membrane potential (JC-1) staining were performed to evaluate mitochondrial biological function. Assays of flow cytometry, reactive oxygen species (ROS) staining, western blot (WB), RT-qPCR, malondialdehyde (MDA), glutathione (GSH), and total superoxide dismutase (SOD) were performed to detect cell ferroptosis, macrophage polarization, and impaired autophagy. The involvement of the P53 pathway was revealed by WB. The results showed that LPS (30 µg/mL) could induce ferroptosis, M1 polarization, mitochondrial dysfunction, and autophagy damage in RAW264.7 cells. Meanwhile, the expression of AQP1 was increased and the expression of P53 was decreased. In addition, Pifithrin-α (PIF; 15 µM), a P53 inhibitor, significantly aggravated ferroptosis, M1 polarization, mitochondrial dysfunction, and autophagy damage as well as up-regulation of AQP1 protein expression in LPS-induced RAW264.7 cells. Interestingly, this phenomenon was markedly alleviated by Kevetrin hydrochloride (70 µM), a P53 agonist. Mechanistically, silencing AQP1 significantly alleviated ferroptosis, M1 polarization, mitochondrial dysfunction, and autophagy damage by up-regulating the expression of P53 in LPS-stimulated RAW264.7 cells. Indeed, inhibition of P53 expression by PIF treatment dramatically reversed this effect on the basis of LPS+si-AQP1. Therefore, we concluded for the first time that AQP1 can promote ferroptosis, M1 polarization, mitochondrial dysfunction, and autophagy impairment by inhibiting the expression of P53 in LPS-stimulated RAW264.7 cells, and AQP1 or P53 may be considered as a crucial determiner that can regulate the biological behavior of RAW264.7 cells stimulated by LPS.

Antioxidant and in vitro cosmeceutical activities of chestnut inner shell fermented by Monascus kaoliang.

Chestnut inner shell (CIS) was fermented at 30 °C for 12 day using Monascus kaoliang, either in solid or submerged state, and alcohol extracts (70% ethanol) of the fermented CIS were examined for their antioxidant (total phenol content and diphenylpicrylhydrazyl radical scavenging activity) and in vitro cosmeceutical activities (tyrosinase and elastase inhibitory activities). Both activities were significantly increased by the M. kaoliang-fermentation, more apparently by submerged fermentation (SMF) than by solid-state fermentation (SSF). The cosmeceutical activity reached its maximum value on the 3rd day of fermentation. The residual amounts of phenolic acids and catechins in the CIS extracts were increased by the fermentation, up to 395.0 and 344.3 µg/g, respectively. More phenolic acids were produced by SMF than SSF, whereas more catechins were produced by SSF than SMF. Therefore, SMF using M. kaoliang was an efficient process for the utilization of CIS as a source of cosmeceuticals.

Analysis of the effectiveness of hearing solutions for congenital malformation of the middle and outer ear (CMMOE).

BACKGROUND: There are no reports about comprehensive comparative analysis of the effects after various hearing surgery solutions for congenital malformation of the middle and outer ear (CMMOE). AIMS/OBJECTIVES: To analyze the improvement of Average Air-Conduction Threshold (AACT) of pure tone after various hearing surgery solutions for CMMOE and provide a reference for the selection of accurate hearing solutions. MATERIALS AND METHODS: A retrospective analysis of 159 cases (170 ears) with CMMOE submitted to various ear surgery solutions, including: (1) Three situations of outer ear canal (OEC): ① atresia 85 ears, ② stenosis 28 ears, and ③ normal 57 ears. (2) Three commonly used hearing solutions: eardrum repair 53 ears, Porp 44 ears and Piston 32 ears implantation. (3) Three OEC situations with different hearing solutions: type I. Reconstruction of OEC (r-OEC), type II. r-OEC and/or different tympanoplasty, including ① eardrum repair, ② release of ossicular chain, ③ Porp implantation, and ④ Torp implantation, type III. Piston implantation with fenestration of the inner ear. Compare AACT of postoperative short term (0.5 years) or long term (0.5-10 years) and preoperative in the speech frequency range of 0.5-4 kHz to assess efficacy. If the sample number ≥10, and not subject to normal distribution, the Kruskal-Wallis multi-sample rank sum test is used for the comparison of multiple groups and Wilcoxon's rank sum test for two groups, with P < 0.05 being statistically significant. If the sample size <10, the standard of clinical efficacy is one frequency improvement value ≥15 dB HL, or 10 dB HL ≤2 frequency improvements <15 dB HL at 0.125-8 KHz. RESULTS: Intra-group comparison of AACT: (1) three situations of OEC: atresia, stenosis and normal all had P < 0.05 postoperatively in short term, while in long term only the normal group had P < 0.05. (2) Three commonly used hearing solutions: eardrum repair, Porp and Piston implantation all had P < 0.05 in short and long terms, except for eardrum repair P >0 .05 in long term. (3) Three OEC situations with different hearing solutions: 1) Atresia of OEC: Porp and Piston implantation, r-OEC and release of ossicular chain were effective in short term and were not effective in long term, and the eardrum repair was not effective in both short and long term. 2) Stenosis of OEC: eardrum repair, Porp and Piston implantation were effective in short and long term. r-OEC P >0 .05 for short and long term, Torp implantation was not effective in long term, 3) Normal of OEC: Porp, Torp and Piston implantation were all P < 0.05 in short and long term except for Torp >0.05 in long term, and release of ossicular chain is both short and long term clinically effective. The AACT values of postoperative in long term for three groups of atresia, stenosis, normal of OEC are over 58.7 dB HL (except Porp implantation 52.5 dB HL), 51.3 dB HL (except Porp implantation 42.5 dB HL), and 37.5 dB HL (except Torp implantation are 32.6 dB HL), respectively. CONCLUSIONS AND SIGNIFICANCE: Intra-group comparison of AACT. (1) Three groups of the atresia, stenosis and normal of OEC are all effective in short term, while in long term only the normal group is effective. (2) The three most commonly used surgical solutions of eardrum repair, Porp and Piston implantation are effective in short and long terms, except for long term eardrum repair. (3) Three OEC situations with different hearing solutions: some of surgical solutions were effective in short term or long term for CMMOE, but based on the AACT values of postoperative in long term for three OEC situations, it is better to choose a hearing device for atresia of OEC, comprehensive review of surgical or hearing device for stenosis of OEC. Surgery can be considered for normal OEC.

[Ortner's syndrome caused by intramural aortic hematoma: a case report].

The clinical manifestations were hoarseness, and no sore throat, fever, dyspnea and dysphagia were found. The patient was in good health and had no history of cardiovascular disease, throat disease, diabetes, asthma, tumor, etc. The left vocal cord paralysis was seen by electronic laryngoscope, and aortic intramural hematoma was found by chest CT examination. In addition, we combined MIMICS digital 3D reconstruction technology to further clarify the lesion. After a series of physical examinations and related examinations, the patient was finally diagnosed as Ortner's syndrome caused by a rare cause of aortic intramural hematoma.

Aquaporin 1 alleviates acute kidney injury via PI3K-mediated macrophage M2 polarization.

BACKGROUND: Lipopolysaccharide (LPS)-induced acute kidney injury (AKI) is associated with an abnormal immune response. Accumulating evidence has demonstrated that aquaporin 1 (AQP1) prevents kidney tissue injury in LPS-induced AKI by mediating immune response. However, the underlying mechanisms remain obscure. Macrophages as immune cells with multiple phenotypes are important mediators in tissue homeostasis and host defense. We propose that macrophage polarization is implicated in AQP1-mediated immune response. METHODS: Herein we established sepsis-induced AKI model rats through intraperitoneal injection of LPS into Wistar rats to reveal immune mechanism of damage. We also used LPS-induced mouse RAW264.7 cells to elucidate the molecular mechanism of macropage polarization. RESULTS: Histopathology showed that renal tubular epithelial cells in the model group were swollen, inflammatory exudation was obvious and the inflammatory factors, interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) were increased. Western blotting showed PI3K was upregulated in the model group. Serum creatinine and urea nitrogen increased after LPS injection. Renal AQP1 mRNA is downregulated and serum AQP1 protein increased first and then decreased in LPS-induced AKI rats. M2 macrophage markers (Arg-1, CD206) were increased in repair stage. In addition, treatment of murine macrophages (RAW264.7) with AQP1 siRNA resulted in decreased PI3K activation and M2 polarization, but increased IL-6 and TNF-α. Moreover, inhibiting PI3K with wortmannin imitated the results of AQP1 silencing. CONCLUSIONS: Macrophage M2 polarization is likely the cellular mechanism underlying the anti-AKI property of AQP1, and PI3K activation is involved in the AQP1-induced M2 phenotype switch.

Aquaporin-1 attenuates macrophage-mediated inflammatory responses by inhibiting p38 mitogen-activated protein kinase activation in lipopolysaccharide-induced acute kidney injury.

OBJECTIVE: This study was designed to investigate the role of AQP1 in the development of LPS-induced AKI and its potential regulatory mechanisms in the inflammatory responses of macrophages. METHODS: Male Wistar rats were injected intraperitoneally with LPS, and biochemical and histological renal damage was assessed. The levels of inflammatory mediators, macrophage markers and AQP1 in blood and kidney tissues were assessed by ELISA. RTPCR was used to assess changes in the relative levels of AQP1 mRNA induced by LPS. Western blot and immunofluorescence analyses were performed to assay the activation of the p38 MAPK and NF-κB pathways, respectively. The same detection methods were used in vitro to determine the regulatory mechanisms underlying AQP1 function. RESULTS: AQP1 mRNA levels were dramatically decreased in AKI rats following the increased expression of inflammatory factors. In vitro experiments demonstrated that silencing the AQP1 gene increased inflammatory mediator secretion, altered the classical activation of macrophages, greatly enhanced the phosphorylation of p38 and accelerated the translocation of NF-κB. Furthermore, these results were blocked by doramapimod, a p38 inhibitor. Therefore, these effects were mediated by the increased phosphorylation of p38 MAPK. CONCLUSION: Our results suggest that altered AQP1 expression may be associated with the development of inflammation in AKI. AQP1 plays a protective role in modulating acute renal injury and can attenuate macrophage-mediated inflammatory responses by downregulating p38 MAPK activity in LPS-induced RAW264.7 cells. The pharmacological targeting of AQP1-mediated p38 MAPK signalling may provide a novel treatment approach for AKI.

Rufomycin Targets ClpC1 Proteolysis in Mycobacterium tuberculosis and M. abscessus.

ClpC1 is an emerging new target for the treatment of Mycobacterium tuberculosis infections, and several cyclic peptides (ecumicin, cyclomarin A, and lassomycin) are known to act on this target. This study identified another group of peptides, the rufomycins (RUFs), as bactericidal to M. tuberculosis through the inhibition of ClpC1 and subsequent modulation of protein degradation of intracellular proteins. Rufomycin I (RUFI) was found to be a potent and selective lead compound for both M. tuberculosis (MIC, 0.02 µM) and Mycobacterium abscessus (MIC, 0.4 µM). Spontaneously generated mutants resistant to RUFI involved seven unique single nucleotide polymorphism (SNP) mutations at three distinct codons within the N-terminal domain of clpC1 (V13, H77, and F80). RUFI also significantly decreased the proteolytic capabilities of the ClpC1/P1/P2 complex to degrade casein, while having no significant effect on the ATPase activity of ClpC1. This represents a marked difference from ecumicin, which inhibits ClpC1 proteolysis but stimulates the ATPase activity, thereby providing evidence that although these peptides share ClpC1 as a macromolecular target, their downstream effects are distinct, likely due to differences in binding.

Inhibition of inflammation using diacerein markedly improved renal function in endotoxemic acute kidney injured mice.

BACKGROUND: Inflammation is an important pathogenic component of endotoxemia-induced acute kidney injury (AKI), finally resulting in renal failure. Diacerein is an interleukin-1ß (IL-1ß) inhibitor used for osteoarthritis treatment by exerting anti-inflammatory effects. This study aims to investigate the effects of diacerein on endotoxemia-induced AKI. METHODS: Male C57BL/6 mice were intraperitoneally injected with lipopolysaccharide (LPS, 10 mg/kg) for 24 h prior to diacerein treatment (15 mg/kg/day) for another 48 h. Mice were examined by histological, molecular and biochemical approaches. RESULTS: LPS administration showed a time-dependent increase of IL-1ß expression and secretion in kidney tissues. Diacerein treatment normalized urine volume and osmolarity, reduced blood urea nitrogen (BUN), fractional excretion of sodium (FENa), serum creatinine and osmolarity, and protected renal function in an endotoxemic AKI mice model. In the histopathologic study, diacerein also improved renal tubular damage such as necrosis of the tubular segment. Moreover, diacerein inhibited LPS-induced increase of inflammatory cytokines, such as IL-1ß, tumor necrosis factor-α, monocyte chemoattractant protein-1 and nitric oxide synthase 2. In addition, LPS administration markedly decreased aquaporin 1 (AQP1), AQP2, AQP3, Na,K-ATPase α1, apical type 3 Na/H exchanger and Na-K-2Cl cotransporter expression in the kidney, which was reversed by diacerein treatment. We also found that diacerein or IL-1ß inhibition prevented the secretion of inflammatory cytokines and the decrease of AQP and sodium transporter expression induced by LPS in HK-2 cells. CONCLUSION: Our study demonstrates for the first time that diacerein improves renal function efficiently in endotoxemic AKI mice by suppressing inflammation and altering tubular water and sodium handing. These results suggest that diacerein may be a novel therapeutic agent for the treatment of endotoxemic AKI.

Cytoprotective effect of aquaporin 1 against lipopolysaccharide-induced apoptosis and inflammation of renal epithelial HK-2 cells.

Sepsis is the most common underlying disease of disseminated intravascular coagulation. Acute kidney injury is a common and serious complications of sepsis. In the present study, a lipopolysaccharide (LPS)-induced human proximal tubule cell line (HK-2 cells) was selected as an in vitro model of septic acute kidney injury. The aim of the present study was to investigate whether aquaporin 1 (AQP-1) has a cytoprotective role in LPS-induced HK-2 cells. HK-2 cells were treated with 0-16 µg/ml LPS for 0-24 h to establish the in vitro model of sepsis. The results demonstrated that AQP-1 levels were the lowest of the eight AQP genes expressed in LPS-induced HK-2 cells. Prior to LPS treatment, HK-2 cells were transfected with pcDNA-AQP-1 or small interfering-AQP-1 and cell counting kint-8 and flow cytometry assays were performed to assess cell viability and apoptosis rate, respectively. Changes in the expression of proinflammatory cytokines and chemokines, as well as important factors in the p38, extracellular signal-regulated kinase (ERK)1/2 and c-Jun N-terminal kinase (JNK) pathways, were assessed using reverse transcription-quantitative polymerase chain reaction, western blotting and ELISA, respectively. LPS treatment reduced viability, increased apoptosis and upregulated the expression of proinflammatory cytokines and chemokines in HK-2 cells. AQP-1 overexpression significantly reversed the effects of LPS and downregulated the expression of tumor necrosis factor-α, interleukin (IL)-8, IL-1ß and monocyte chemoattractant protein-1. The p38, ERK1/2 and JNK pathways were activated by LPS; however, the p38 and ERK1/2 pathways were blocked in AQP-1-overexpressing cells. AQP-1 overexpression was demonstrated to confer a survival advantage to LPS-injured HK-2 cells by controlling cell viability, apoptosis and inflammation, possibly via modulation of the p38 and ERK1/2 pathways. The results of the present study suggest that AQP-1 may be an effective treatment for acute kidney injury caused by sepsis.

Discovery and characterization of the tuberculosis drug lead ecumicin.

The new tuberculosis (TB) lead ecumicin (1), a cyclic tridecapeptide, was isolated from Nonomuraea sp. MJM5123, following a high-throughput campaign for anti-TB activity. The large molecular weight of 1599 amu detected by LC-HR-MS precluded the initial inference of its molecular formula. The individual building blocks were identified by extensive NMR experiments. The resulting two possible planar structures were distinguished by LC-MS(2). Determination of absolute configuration and unambiguous structural confirmation were carried out by X-ray crystallography and Marfey's analysis.

Adenovirus type 3 induces platelet activation in vitro.

In the present study, we aimed to investigate platelet activation induced by adenovirus type 3 (HAdV3) in vitro. Platelet-rich plasma (PRP) or whole blood was incubated with or without HAdV at various concentrations. Platelet aggregation, platelet counting, fibrinogen and expression of platelet membrane antigens (CD41a and CD62P) were determined following incubation with HAdV for different periods of time. The results demonstrated that HAdV at the concentrations of 109-1011 vp/ml enhanced adenosine diphosphate (ADP) or ristocetin-induced platelet aggregation, however did not alter the platelet count. Infection with HAdVs also reduced fibrinogen level. P-selectin and CD41a appeared rapidly on the surface after platelets were incubated with HAdVs in vitro for 30 min. In conclusion, HAdVs may induce activation of platelets and lead to a pre-thrombotic state of peripheral blood. This finding may aid in the development of measures to prevent severe HAdV infection.

Clinical implications of CIP2A protein expression in breast cancer.

The aim of this work is to study the expression and regulatory effects of CIP2A protein in breast cancer and the correlation between CIP2A protein expression and the prognosis of breast cancer. The CIP2A protein level was detected by immunohistochemistry staining. The relationship between CIP2A protein and clinicopathological parameters of breast cancer was determined. It was observed that 448 (35.00 %) of the 1,280 cases positively expressed CIP2A protein. Univariate analysis indicated that CIP2A expression was related to histological grade, lymph node metastasis, distant metastasis, and triple-negative breast cancer (P = 0.001, 0.001, 0.001, and 0.001, respectively). Spearman correlation analysis showed that CIP2A expression has line correlation with histological grade, lymph node metastasis, distant metastasis, triple-negative breast cancer, and TNM stage (P = 0.03, 0.001, 0.008, 0.001, and 0.001, respectively). After multivariate analysis, tumor size, histological grade, lymph node metastasis, triple-negative breast cancer, distant metastasis, and TNM stage were related to CIP2A expression (P = 0.035, 0.001, 0.028, 0.001, 0.001, and 0.001, respectively). CIP2A expression also significantly related to chemotherapeutic sensitivity of breast cancer in the neoadjuvant chemotherapy. In the Cox regression test, histological grade, lymph node metastasis, triple-negative breast cancer, and TNM stage were detected as the independent prognostic factors (P = 0.001, 0.006, 0.01, 0.011, and 0.001, respectively). CIP2A expression may be a potential biomarker for chemotherapeutic sensitivity and prognosis of breast cancer.

S-adenosyl-L-methionine activates actinorhodin biosynthesis by increasing autophosphorylation of the Ser/Thr protein kinase AfsK in Streptomyces coelicolor A3(2).

S-Adenosyl-L-methionine (SAM) is one of the major methyl donors in all living organisms. The exogenous treatment with SAM leads to increased actinorhodin production in Streptomyces coelicolor A3(2). In this study, mutants from different stages of the AfsK-AfsR signal transduction cascade were used to test the possible target of SAM. SAM had no significant effect on actinorhodin production in afsK, afsR, afsS, or actII-open reading frame 4 (ORF4) mutant. This confirms that afsK plays a critical role in delivering the signal generated by exogenous SAM. The afsK-pHJL-KN mutant did not respond to SAM, suggesting the involvement of the C-terminal of AfsK in binding with SAM. SAM increased the in vitro autophosphorylation of kinase AfsK in a dose-dependent manner, and also abolished the effect of decreased actinorhodin production by a Ser/Thr kinase inhibitor, K252a. In sum, our results suggest that SAM activates actinorhodin biosynthesis in S. coelicolor M130 by increasing the phosphorylation of protein kinase AfsK.

Combination of small interfering RNAs mediates greater suppression on hepatitis B virus cccDNA in HepG2.2.15 cells.

AIM: To observe the inhibition of hepatitis B virus (HBV) replication and expression in HepG2.2.15 cells by combination of small interfering RNAs (siRNAs). METHODS: Recombinant plasmid psil-HBV was constructed and transfected into HepG2.2.15 cells. At 48 h, 72 h and 96 h after transfection, culture media were collected and cells were harvested for HBV replication assay. HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay (ELISA). Intracellular viral DNA and covalently closed circular DNA (cccDNA) were quantified by real-time polymerase chain reaction (PCR). HBV viral mRNA was reverse transcribed and quantified by reverse-transcript PCR (RT-PCR). RESULTS: siRNAs showed marked anti-HBV effects. siRNAs could specifically inhibit the expression of HBsAg and the replication of HBV DNA in a dose-dependent manner. Furthermore, combination of siRNAs, compared with individual use of each siRNA, exerted a stronger inhibition on antigen expression and viral replication. More importantly, combination of siRNAs significantly suppressed HBV cccDNA amplification. CONCLUSION: Combination of siRNAs mediates a stronger inhibition on viral replication and antigen expression in HepG2.2.15 cells, especially on cccDNA amplification.

Combination therapy of siRNAs mediates greater suppression on hepatitis B virus cccDNA in HepG2.2.15 cell.

BACKGROUND/AIMS: Hepatitis B virus (HBV) infection is a world-wide health problem. The major obstacles for current anti-HBV therapy are the low efficacy and the occurrence of drug resistant HBV mutations. Recent studies have demonstrated that combination therapy can enhance antiviral efficacy and overcome the shortcomings. Here, the inhibitory effect mediated by combination of small interfering RNAs (siRNAs) targeting different sites of HBV nuclear localization signal (NLS) was monitored in HepG2.2.15 cells. METHODOLOGY: Recombinant plasmid psil-HBV was constructed and transfected into HepG2.2.15 cells. At 48, 72 and 96 h after transfection, culture media were collected and cells were harvested for HBV replication assay. HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay. Intracellular viral DNA and covalently closed circular DNA (cccDNA) was quantified by real-time PCR. HBV viral mRNA was reverse transcribed and quantified by reverse-transcript PCR. RESULTS: Our data demonstrated that three used siRNAs showed marked anti-HBV effects. The expression of HBsAg and the replication of HBV DNA could be specifically inhibited in a dose-dependent manner by siRNAs. Furthermore, combination of siRNAs, compared with individual use of each siRNA, exerted a stronger inhibition on antigen expression and viral replication, even though the final concentration of siRNA in the therapy was the same. More importantly, we showed that combination therapy significantly suppressed HBV cccDNA amplification. CONCLUSION: Our results revealed that combination of siRNAs mediated a stronger inhibition on viral replication and antigen expression in HepG2.2.15 cells, especially, the amplification of cccDNA.