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BackgroundA recently developing pneumonia caused by SARS-CoV-2 was originated in Wuhan, China, and has quickly spread across the world. We reported the clinical characteristics of 82 death cases with COVID-19 in a single center.MethodsClinical data on 82 death cases laboratory-confirmed as SARS-CoV-2 infection were obtained from a Wuhan local hospital’s electronic medical records according to previously designed standardized data collection forms.ResultsAll patients were local residents of Wuhan, and the great proportion of them were diagnosed as severe illness when admitted. Most of the death cases were male (65.9%). More than half of dead patients were older than 60 years (80.5%) and the median age was 72.5 years. The bulk of death cases had comorbidity (76.8%), including hypertension (56.1%), heart disease (20.7%), diabetes (18.3%), cerebrovascular disease (12.2%), and cancer (7.3%). Respiratory failure remained the leading cause of death (69.5%), following by sepsis syndrome/MOF (28.0%), cardiac failure (14.6%), hemorrhage (6.1%), and renal failure (3.7%). Furthermore, respiratory, cardiac, hemorrhage, hepatic, and renal damage were found in 100%, 89%, 80.5%, 78.0%, and 31.7% of patients, respectively. On the admission, lymphopenia (89.2%), neutrophilia (74.3%), and thrombocytopenia (24.3%) were usually observed. Most patients had a high neutrophil-to-lymphocyte ratio of >5 (94.5%), high systemic immune-inflammation index of >500 (89.2%), increased C-reactive protein level (100%), lactate dehydrogenase (93.2%), and D-dimer (97.1%). A high level of IL-6 (>10 pg/ml) was observed in all detected patients. Median time from initial symptom to death was 15 days (IQR 11-20), and a significant association between aspartate aminotransferase (p=0.002), alanine aminotransferase (p=0.037) and time from initial symptom to death were interestingly observed.ConclusionOlder males with comorbidities are more likely to develop severe disease, even die from SARS-CoV-2 infection. Respiratory failure is the main cause of COVID-19, but either virus itself or cytokine release storm mediated damage to other organ including cardiac, renal, hepatic, and hemorrhage should be taken seriously as well.FundingNo founding.Research in contextEvidence before this studyAs the seventh member of enveloped RNA coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV)-2 causes a cluster of severe respiratory disease which is similar to another two fatal coronavirus infection caused by SARS-CoV and Middle Eastern respiratory syndrome coronavirus (MERS-CoV). Through searching PubMed and the China National knowledge infrastructure databases up to February 20, 2020, no published article focusing on hospitalized dead patients was identified.Added value of this studyWe conducted a single-center investigation involving 82 hospitalized death patients with COVID-19 and focused on their epidemiological and clinical characteristics. 66 of 82 (80.5%) of patients were older than 60 years and the median age was 72.5 years.The bulk of death cases had comorbidity (76.8%). Respiratory failure remained the leading cause of death, following by sepsis syndrome/MOF, cardiac failure, hemorrhage, and renal failure. Most patients had a high neutrophil-to-lymphocyte ratio, high systemic immune-inflammation index, and increased levels of proinflammatory cytokines.Implications of all the available evidenceSARS-CoV-2 causes a cluster of severe respiratory illness which is similar to another two fatal coronavirus infection caused by SARS-CoV and MERS-CoV. Death is more likely to occur in older male patients with comorbidity. Infected patients might develop acute respiratory distress and respiratory failure which was the leading cause of death, but damages of other organs and systems, including cardiac, hemorrhage, hepatic, and renal also contribute to the death. These damages might be attributable to indirect cytokines storm initiated by immune system and direct attack from SARS-CoV-2 itself.

To improve the quality of cultured large yellow croaker (Larimichthys crocea), this study was performed to study the impacts of glycerol monolaurate (GML) on the nutritional value, growth performance, muscle texture, and taste intensity of L. crocea. The results showed that GML as a feed additive significantly increased the crude lipid content and reduced the diameters of muscle fibers, which in turn markedly altered the flesh texture in terms of cohesiveness. Moreover, the taste indicators (umami and richness) and flavor-related amino acid (glutamic acid, glycine, and proline) contents of L. crocea muscle were significantly higher in the GML group. Metabolomic and gene expression analyses showed that GML supplementation could significantly improve amino acid biosynthesis and metabolism, promote protein and lipid synthesis, and activate myogenic-related signaling pathways of L. crocea. Consequently, adding an appropriate amount of GML to fish feed would be conducive to providing healthy, nutrient-rich and acceptably flavored aquatic-products.

BACKGROUND Dialysis frequency and dose are controversial prognostic factors of hemodialysis morbidity and mortality. The aim of this study was to find out the effect of frequency and dosage of dialysis on mortality and survival in a group of Chinese hemodialysis patients. MATERIAL AND METHODS In total, 183 patients seen from February 2008 to January 2018, who were on maintenance hemodialysis for at least 3 months, were included in the study cohort. An anonymized database of age, gender, diabetic status, comorbidities, date of initiation of dialysis, hematological characters, biochemical variables, and status of survived or died was established from DICOM (Digital Imaging and Communications in Medicine) files of patients. Kaplan-Meier and Cox-proportional hazard model was used for calculation of survival over time at 95% confidence level. RESULTS Overall, the 10-year survival rate was 27%. Kaplan-Meier analysis showed patient survival as 94% at one-year, 59% at 5-years, and 27% at 10-years. Hemoglobin, serum albumin, calcium, potassium, phosphorous, calcium-phosphorous-products, and hemodialysis frequency and the dose had a significant effect on survival. Cox regression proportional hazard model showed that patients with serum albumin level of >4 g/dL were better associated with survival. Patients who underwent twice-weekly hemodialysis had 4.26 times less chance of survival as compared to patients with thrice-weekly hemodialysis. A higher dialysis dose of >1.2 spKt/V offered better survival as compared to a lower dose of

Land-based recirculating aquaculture systems (RAS) and cage culture are important methods of Larimichthys crocea production. The effects of environmental factors on physiological and biochemical aspects of L. crocea require clarification. Temperature and salinity are controlled in RAS and directly affect L. crocea growth and survival. To explore optimal parameters, the oxygen consumption rate (R O ), ammonium excretion rate (R N ), and O/N ratio at different temperatures (8, 14, 20, 26, and 32 °C) and salinities (5, 15, 25, and 35‰) were determined. R O , R N , and O/N first increased and then decreased with elevated temperature and salinity, peaking at 26 °C and 25‰, respectively. This suggests that the metabolism of L. crocea was maximal at 26 °C and 25‰ salinity, which promote its growth and survival. Additionally, hypoxia affects cage culture, and has significant effects on enzymatic activities and stress-inducible gene expression. To accelerate the selective breeding of hypoxia-tolerant L. crocea in cage culture, we measured adenosine triphosphatase (ATPase), lactate dehydrogenase (LDH), and succinate dehydrogenase (SDH) activities, and hypoxia-inducing factor 1 (HIF-1) mRNA expression in the myocardium under hypoxia (2.5, 3.5, and 4.5 mg L −1 ). ATPase and SDH activities first decreased and then increased under hypoxia, whereas LDH activity and HIF-1α expression first increased and then decreased. Thus, under hypoxia, the myocardial mitochondria switched from being susceptible to being resistant to injury induced by energy metabolism, and respiration in L. crocea likely converted from aerobic to anaerobic during adaptation. Furthermore, the upregulation of HIF-1α mRNA suggests it has an active role in protection against anoxic damage.

Organisms at all levels of evolutionary complexity react to hypoxic stress. To clarify the effects of acute hypoxia on physiological and biochemical responses of Larimichthys crocea, we measured the activity levels of the antioxidant enzymes superoxide dismutase and catalase, hemoglobin concentration, functional indices of the liver (aspartate transaminase, alanine transaminase), heart (phosphocreatine kinase), and immune system (alkaline phosphatase), as well as mRNA expression levels of the immunity-related genes Hsp70 and HIF-1α at different time points of hypoxic. In addition, liver, gill, and kidney samples were histologically analyzed. We found that hemoglobin concentration and all enzyme activities increased during hypoxia, although these effects were transient and most indices returned to basal levels thereafter. The extent of the increase in the parameter values was inversely proportional to the dissolved oxygen content. Hsp70 and HIF-1α mRNA expression levels increased significantly in the blood, liver, gills, and kidneys following exposure to hypoxia, which may play an important role in protecting fish against oxidative damage. However, we found histological evidence of hypoxia-induced injuries to the gills, liver, and kidneys, which are involved in breathing, detoxification, and osmotic balance maintenance, respectively. Thus, despite the upregulation of defensive mechanisms, acute hypoxia still caused irreversible damage of organs. In conclusion, we observed that, in response to acute hypoxic stress, L. crocea enhances immune defensive function and antioxidant capacity. A better understanding of the regulation of the molecular anti-hypoxia mechanisms can help speeding up the selective breeding of hypoxia-tolerant L. crocea.

Tristetraprolin (TTP) regulates inflammatory and immune responses by destabilizing target mRNAs via binding to their 3'-UTR AREs. We have recently reported that TTP preferentially up-regulates the expression level of innate immunity genes involved in the type I interferon-mediated signaling pathway and viral response in cancer cells. To elucidate the role of TTP-RNA interaction in TTP-mediated upregulation of gene expression, we performed iRIP-seq experiments to obtain the RNA interaction map consisting of direct and indirect binding sites of TTP in HeLa cells. We found substantial TTP binding signals in mRNA regions and the introns. ARE-motif AUUUA is over-represented in TTP binding peaks. Strikingly, AUUUA frequency is high both in 3'UTR and intronic regions, and the intronic peaks were more associated with TTP-regulated genes. Analysis of the over-represented motifs in TTP peaks revealed the high frequencies of UAGG and GUGUG motifs reported for hnRNPA2/B1 and CELF1 respectively in the 3'UTR and introns, and also the UGGAC motif overlapping with the m6A motif GGACU in the CDS regions. We further demonstrated that TTP binds to multiple intronic and exonic sites in the pre-mRNA/mRNA of the transcription factor RelB, correlating with the TTP-upregulated expression of RelB. TTP-up-regulated genes without a TTP binding site, but not those with, are highly enriched in innate immunity pathways and show higher tendency of harboring RelB binding sites in their promoter regions. These findings support a model in which TTP binding of RelB pre-mRNA/mRNA coordinates the RelB upregulation and activation of the innate immunity for antiviral response.

Low temperature influences multiple physiological processes in fish. To explore the adaptability of the large yellow croaker (Larimichthys crocea) to low temperature, the concentrations of glycerol, blood urea nitrogen (BUN), and triglycerides (TG) in plasma, as well as the expression levels of metabolism-related genes aqp7 and aqp10, were measured after exposure to low temperature stress and during subsequent rewarming. In addition, tissue samples from the intestine and liver were histologically analyzed. We found that the concentrations of plasma glycerol, BUN, and TG, decreased under low temperature stress, suggesting the metabolism of fat and protein slowed at low temperature. The expression levels of aqp7 and aqp10 mRNA were also downregulated under exposure to low temperature. Interestingly, above plasma indices and gene expression returned to basic levels within 24 h after rewarming. Furthermore, the liver and the intestine were damaged under continuous low temperature stress, whereas they were repaired upon rewarming. From the above results, we concluded that aqp7 and aqp10 genes were sensitive to low temperature, and that the decrease in their expression levels at low temperature might reduce energy consumption by L. crocea. However, the adaptation to low temperature was limited because the key metabolic tissues were damaged under continuous exposure to low temperature. Interestingly, the metabolism of L. crocea was basically back to normal within 24 h of rewarming, showing that it has high capacity of self-recovery.

Supporting figures and tables. (DOCX 378 kb)

A 10-week feeding trial was conducted to evaluate the effect of dietary methionine (MET) on the growth performance, lipid metabolism and liver antioxidant function in juvenile large yellow croaker (Larimichthys crocea) fed with high lipid diets (HLDs). Large yellow croaker with an initial average weight of 8.59 g, were fed with basic HLD (lipid level at 18%, HL), basic HLD supplemented with 0.6% MET (HL/L-MET) and basic HLD supplemented with 1.2% MET (HL/H-MET). Results indicated that dietary MET did not affect weight gain, specific growth rate and feed efficiency ratio (P > 0.05). The MET supplementation in large yellow croaker also did not affect the content of total lipid in whole body (HL = 9.57, HL/L-MET = 8.92 and HL/H-MET = 8.71%), liver (7.34, 16.68 and 14.37%) and muscle (10.87, 12.11 and 9.94%) (P > 0.05). However, MET supplementation reduced the size and number of liver lipid droplets as dietary MET increased. Meanwhile, supplementation of MET in HLDs reduced serum total cholesterol (2.65, 2.14 and 1.91 mmol/L), low-density lipoprotein-cholesterol (0.27, 0.21 and 0.16 mmol/L) and triglyceride (4.80, 2.43 and 1.96 mmol/L) (P

To explore the effect of environment conditions on immune activity of fish, eight immune-associated genes responsible for innate immunity were selected from the GenBank, i.e. Pgrn-a, Ifit2, P-hepcidin, Lect2, β2m, Irf1, Il25 and Hsp96, and the mRNA expressions of them in the kidney of cultured large yellow croaker Larimichthys crocea in different sea areas in the East China Sea were examined with qPCR techniques. In the contrasts of immune-associated gene expression between areas and populations, significant differences were found, expression levels of these immune-associated genes were lower in the clear water area than in the poor water quantity area, and lower in May than in October. MY was more sensitive to environmental factors than DQ, which was coincident with the water quality in the culturing areas. Differential analyses of the expression levels of these immune-associated genes showed that significant up-regulation could be triggered by poor environmental factors. The expression patterns indicated that the expression levels of these genes were sensitive to ecological changes, thereby the immune-associated genes, especially Pgrn-a, Ifit2, β2m, Il25 and Hsp96, might serve as immediate and sensitive indicators of population immunologic vigor and ecosystem health. But the expression of immunity-associated genes at the level of gene transcription is highly influenced by multiple factors, and the exact causes or influencing factors of the up-regulation or down-regulation of these genes still need further thorough investigation.

BACKGROUND The aim of this study was to explore the potential role of TRPC6 in the pathophysiology of HK-2 cell injury following ischemia reperfusion (I/R). MATERIAL AND METHODS TRPC6 expression was analyzed by immunofluorescence staining. siRNA was transfected to knockout of TRPC6 in HK-2 cells, and in vitro I/R was then induced. Cell apoptosis and necrosis were determined by Annexin V-FITC/PI staining. Necroptosis was determined by necrostatin-1 and expressions of necroptosis-related proteins were evaluated. OAG, SKF96365, or KN-93 was further used to interfere with TRPC6 expression. RESULTS Cytoplasmic TRPC6 expression was demonstrated. I/R induced TRPC6 expression in normal or NC siRNA-transfected cells but not in TRPC6 siRNA-knockout ones. There was a progressive increase in apoptotic and necrotic cells with increasing reoxygenation time in all 3 groups, while necrosis in TRPC6 siRNA-transfected cells was comparatively higher than that of the other 2 groups (p

The role of microRNAs (miRNAs) in chronic kidney disease (CKD) is relatively well established, but much less is known about the role(s) of long noncoding RNAs (lncRNAs). Transforming growth factor β1 (TGF-β1) mediates inflammatory and fibrogenic signaling in CKD via the transcription factor Smad3; however, the extent of lncRNAs-based regulation of TGF-β1 signaling in CKD remains unknown. Herein, we identified np_4334, a lncRNA we named Ptprd-IR, whose promoter contains a highly-conserved site for Smad3 binding. Smad3 knockout (KO) eliminated Ptprd-IR upregulation in a murine model of obstructive nephropathy. Furthermore, Ptprd-IR KO in renal tubular epithelial cell cultures blocked TGF-β1- and interleukin-1β (IL-1β)-mediated NF-κB inflammatory signaling but did not impact TGF-β1-triggered Smad3 pathway activity and fibrosis. Accordingly, Ptprd-IR overexpression (OE) upregulated TGF-β1- and IL-1β-mediated NF-κB pathway activation and production of pro-inflammatory cytokines but did not influence TGF-β1-mediated fibrogenic signaling. Additionally, transfection of obstructed kidneys with Ptprd-IR-directed shRNA attenuated the inflammatory response via NF-κB but did not impact TGF-β1/Smad3-mediated fibrogenesis. Overall, our findings demonstrate that the lncRNA Ptprd-IR stimulates the inflammatory response in kidneys and advocate Ptprd-IR as a possible therapeutic target for CKD.

The large yellow croaker (Larimichthys crocea) is one of the most important marine economic fish in the southeast coast of China. However, hypoxia stress become a major obstacle to the benign development of L. crocea industry. To understand the energy metabolism mechanism adapted to hypoxia, we analyzed the transcriptome and physiology of L. crocea liver in response to hypoxia stress for different durations. We obtained 243,756,080 clean reads, of which 83.38% were successfully mapped to the reference genome of L. crocea. The heat map analysis showed that genes encoding enzymes involved in glycolysis/gluconeogenesis were significantly upregulated at various time points. Moreover, genes encoding enzymes related to the citrate cycle, oxidative phosphorylation, and amino acid metabolism were significantly downregulated at 6 and 24 h, but upregulated at 48 and 96 h. The change of liver in physiology processes, including respiratory metabolism, and activities of the carbohydrate metabolism enzymes showed a similar trend. The results revealed that the respiratory metabolism of L. crocea was mainly anaerobic within 24 h of hypoxia stress, and aerobic metabolism was dominant after 24 h. Carbohydrate metabolism plays a crucial role in energy supply and amino acid metabolism is an important supporting character to cope with acute hypoxia stress. There was no significant change in lipid utilization under short-term acute stress. This study increases our understanding of the energy metabolism mechanism of the hypoxia response in fish and provides a useful resource for L. crocea genetics and breeding.

Necroptosis, oxidative stress, and inflammation are major contributors to the pathogenesis of ischemic acute kidney injury. Necrostatin-1 (Nec-1), an inhibitor of the kinase domain of receptor-interacting protein kinase-1 (RIP1), has been reported to regulate renal ischemia and reperfusion (I/R) injury; however, its underlying mechanism of action remains unclear. HK-2 cells were used to create an in vitro I/R model, in which the cells were subjected to hypoxia, followed by 2, 6, and 12 h of reoxygenation. For the in vivo study, a rat model of renal I/R was established in which samples of rat blood serum and kidney tissue were harvested after reperfusion to assess renal function and detect histological changes. Cell viability and necroptosis were analyzed using the Cell Counting Kit (CCK)-8 assay and flow cytometry, respectively. The expression levels of molecules associated with necroptosis, oxidative stress, and inflammation were determined by real-time PCR, western blotting, immunofluorescence staining, and ELISA. Luciferase and chromatin immunoprecipitation (ChIP) assays were performed to confirm the relevant downstream signaling pathway. We found that pretreatment with Nec-1 significantly decreased hypoxia-inducible factor-1α (HIF-1α) and miR-26a expression, as well as the levels of factors associated with necroptosis (RIP1, RIP3, and Sirtuin-2), oxidative stress (malondialdehyde [MDA], NADP+/NADPH ratio), and inflammation (interleukin [IL]-1β, IL-10, and tumor necrosis factor alpha [TNF-α]) in I/R injury cells and the rat model. However, these effects could be reversed by miR-26a overexpression or TRPC6 knockdown. Mechanistic studies demonstrated that HIF-1α directly binds to the promoter region of miR-26a, and that TRPC6 is a potential target gene for miR-26a. Our findings indicate that Nec-1 can effectively protect against renal I/R injury by inhibiting necroptosis, oxidative stress, and inflammation, and may exert its effects through mediation of the HIF-1α/miR-26a/TRPC6/PARP1 signaling pathway. Keywords: ischemic/reperfusion injury, Nec-1, HIF-1α, miR-26a, TRPC6, necroptosis, oxidative stress, inflammation

Human scavenger receptor B type CD36 (CD36), a member of the class B scavenger receptors, has been shown to play a critical role in the formation of atherosclerosis and may be a potential target for the treatment of atherosclerosis. Sensitive and accurate determination of CD36 is strongly desired in clinical testing in recent years. On the basis of the improved sensitivity of electrochemical immunoassays, a novel electrochemical immunosensor was constructed for CD36 with glucose oxidase (GOx) labeled Prussian blue nanoparticle functionalized Ceria nanoparticles (PB@CeO2NPs) as the signal enhancer. The prepared nanohybrid of PB@CeO2NPs exhibits excellent electrochemical catalytic activity like “artificial peroxidase”, with the cooperative catalysis of Prussian blue nanoparticles (PBNPs) and Ceria nanoparticles (CeO2NPs) for the effective reduction of H2O2, as well as strong and stable quasi-reversible cyclic voltammetry curves due to the important roles of PBNPs with their intrinsic redox electrochemical properties. Significantly, the electrochemical signal was greatly enhanced due to cascade catalysis: firstly, GOx catalyzed the deoxidization of glucose to gluconic acid with the concomitant generation of H2O2; then H2O2 was further decomposed by the catalysis of the “artificial peroxidase” of PBNPs and CeO2NPs. Thus, this proposed method was developed for the determination of CD36, based on sandwich-type immunoreactions, and it exhibited good electrochemical responses at a linear calibration range from 5.0 × 10−3 ng mL−1 to 80 ng mL−1, with a detection limit of 2.0 pg mL−1.

Cyp2e1 plays an important role in chemically induced hepatocarcinogenesis. Resveratrol (REV) is known to prevent diethylnitrosamine (DEN)-induced hepatocarcinogenesis, but its effects on this process induced by DEN and 2-acetylaminofluorene (2-AAF) and the role of Cyp2e1 remain unclear. In this study, glutathione S-transferase placental form (GST-P)-positive foci were used as a marker of hepatocarcinogenesis. REV or diallyl disulfide (DADS, an inhibitor of Cyp2e1) significantly reduced both the area and number of GST-P-positive foci induced by DEN and 2-AAF. Treatment with REV or DADS also markedly decreased the expression of Cyp2e1 in the rat liver. By immunohistochemical staining of serial liver sections, we found that the expression of Cyp2e1 in GST-P-positive foci showed three distinct patterns: decreased in GST-P foci, increased in GST-P foci when compared with surrounding liver tissue and mixed type. The number of GST-P foci with increased Cyp2e1 expression was greater than the number of GST-P foci with decreased Cyp2e1. Protein levels of GST-P and Cyp2e1 were also higher in foci compared with surrounding liver tissue. REV or DADS significantly reduced the expression of GST-P and Cyp2e1 in both foci and surrounding liver tissue. Taken together, these results suggested that REV has a significant inhibitory effect on chemically induced hepatocarcinogenesis, which may be attributed to downregulation of Cyp2e1.

Background Regulatory T (Treg) cells play important roles in autoimmune diseases, cancer, and organ transplantation. Forkhead box protein o1 (Foxo1) and IL-7Rα(CD127) are closely related to the homeostasis of Treg cells. However, the mechanism underlying Treg proliferation and activation remains unclear. Here, we evaluated how the over-expression of Foxo1 affects Treg cell proliferation via intracellular signaling. nTreg cells were transfected separately with Foxo1 and Aven small-interfering RNA (siRNA) or over-expression plasmid. The expression of signaling pathway genes and CD127 was confirmed using RT-qPCR and western blot analysis. The expression of cell surface molecules and apoptosis was confirmed by Flow Cytometry 3-(4, 5-Dimethylthiazol-2-yl) 2,5- diphenyltetrazolium bromide for cell proliferation assays. Results Foxo1 strengthened the proliferative ability of Treg cells by activating IL-7/CD127 signaling. In addition, Foxo1 suppressed Treg cell apoptosis by regulating Aven expression. Conclusions The results in this study indicated that Foxo1 is a positive regulatory factor for the proliferation and activity of Treg cells. Foxo1 might be a potential target for the activation of nTreg cells in vivo and in vitro.

A novel electrochemical aptasensor for ultrasensitive detection of thrombin is described that uses the conductive graphene-3,4,9,10-perylenetetracarboxylic dianhydride nanocomposites (GPA) as desirable sensor platform and hollow PtCo nanochains–thionine–Pt–HRP labeled secondary thrombin aptamer (HPtCoNCs–Thi–Pt–HRP–TBA, aptamer II) for signal amplification. With the surface area enhanced by GPA nanocomposites, the amount of immobilized primary thrombin aptamer can be improved. More importantly, the synthesized HPtCoNCs also yielded large surface area, which provide substantial binding sites for thionine–Pt–HRP–TBA. The thionine acts as a mediator of electron transfer between HRP and electrode surface. Enhanced sensitivity was achieved by introducing the multibioconjugates of aptamer II onto the electrode surface through “sandwich” reactions. With amplifying factors mentioned above, a wider linear range from 10 −15 to 10 −9 M was acquired with an ultralow detection limit of 6.5 × 10 −16 M for thrombin. This amplification strategy showed acceptable stability and reproducibility, high sensitivity, and good precision, which could provide a promising potential for clinical screening and point-of-care diagnostics.

National Grand Fundamental Research 973 Program of China [2006CB504700, 2007CB914304]; National Science and Technology Major Project "Key New Drug Creation and Manufacturing Program", China [2009ZX09301-001, 2008ZX09305-007, 2009ZX09501-033]; Program of Shanghai Subject Chief Scientist [09XD1405100]

Introduction Few have tried to prove the effectiveness of mizoribine combined with losartan for adult IgA nephropathy patients in a randomized controlled trial Methods A multicenter, randomized, controlled, 12-month study was performed to evaluated the efficacy and safety of mizoribine combined with losartan for adult IgA nephropathy. Ninety-nine patients with primary IgA nephropathy from 8 clinical institutions were randomly assigned to the losartan group (n = 30), the mizoribine group (n = 35) or the combination (losartan+mizoribine) group (n = 34). The primary outcome was 24-hour urinary protein excretion (24 hours-UP). Results There were no significant differences in baseline data among the 3 groups. In all 3 groups, 24 hours-UP after 3, 6, 9 and 12 months of treatment were significantly lower than the baseline level. The reduction in 24 hours-UP in the losartan group was observed early and reached maximum after 6 months of treatment. Twenty-four hours-UP in the mizoribine group and combination group continuously decreased during the study. Comparisons among the 3 groups showed that the losartan group was superior to the mizoribine group after 3 months of treatment, but that after 12 months of treatment, both the combination group and the mizoribine group were superior to the losartan group in the reduction of 24 hours-UP. There were no significant differences among the 3 groups in serum creatinine. No serious adverse events occurred in any of the 3 groups. Conclusions The treatment of adult IgA nephropathy with mizoribine alone, losartan alone or a combination of the 2 reduced 24 hours-UP. Mizoribine and losartan, when used in combination, complement each other’s activities.

Through electrodepositing Prussian blue (PB) and chitosan (CS), then casting Pt hollow nanospheres (HN-Pt) and assembling CA19-9 antibody on the electrode surface, an immunosensor was achieved. A new signal amplification strategy based on PB and HN-Pt toward the electrocatalytic reduction of H2O2 was employed when performing the determination. The resulting immunosensor showed a high sensitivity, broad linear response to carbohydrate antigen 19-9 (CA19-9) in two ranges from 0.5 to 30 and 30 to 240 U mL−1 with a low detection limit of 0.13 U mL−1 (S/N=3). Moreover, it displayed good reproducibility and stability, and would be potentially attractive for clinical immunoassay of CA19-9.

Centipede venoms are complex mixtures of biochemically and pharmacologically active components such as peptides and proteins. Very few are known about their pharmacological actions. The present work reports the structural and functional characterization of two antimicrobial peptides (scolopin 1 and -2) identified from centipede venoms of Scolopendra subspinipes mutilans by Sephadex gel filtration and reverse-phase high-performance liquid chromatography (RP-HPLC). The amino acid sequences of scolopin 1 and -2 were FLPKMSTKLRVPYRRGTKDYH and GILKKFMLHRGTKVYKMRTLSKRSH determined by Edman degradation and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Both scolopin 1 and -2 showed strong antimicrobial activities against tested microorganisms including Gram-positive/negative bacteria and fungi. They also showed moderate hemolytic activity against both human and rabbit red cells. This is the first report of antimicrobial peptides from centipedes.

VSIG4 has been recently described as a B7 family-related protein. The immunotherapeutic potential of dendritic cells (DCs) transfected with VSIG4 recombinant adenovirus has not been characterized. In the present study, DCs were transfected with human VSIG4 (hVSIG4) recombinant adenovirus, a novel costimulatory molecule known to be a potent inhibitor of T cell activation. Transfected DCs were cocultured with allogeneic T cells and proliferation, cytokine production and T cell activation marker expression were assessed. The results showed that T cell proliferation potential, cytokine production and activation marker expression were suppressed after coculture with hVSIG4 recombinant adenovirus-transfected DCs. These findings suggest that DCs transfected with hVSIG4 recombinant adenovirus are capable of inducing allogeneic T cell suppression, which represents an ideal strategy for manipulating the immune response to transplanation or autoimmune diseases.

B-cell-activating factor (BAFF), a member of the tumour necrosis factor superfamily, plays a critical role in the maturation, homeostasis and function of B cells. In this study, we demonstrated the biological outcome of BAFF blockade in BXSB murine lupus model, using a soluble fusion protein consisting of human BAFF-R and human mutant IgG4 Fc. Mutation of Leu235 to Glu in IgG4 Fc eliminated antibody-dependent cell cytotoxicity (ADCC) and complement lysis activity, and generated a protein devoid of immune effector functions. Treatment of BXSB mice with BAFF-R-IgG4mut fusion protein for 5 weeks resulted in significant B-cell reduction in both the peripheral blood and spleen. Treated mice developed lower proteinuria, reduced glomerulonephritis and much delayed host death than untreated animals. Thus, BAFF blockade with BAFF-R-IgG4mut protein is an effective strategy to treat B-cell-mediated lupus-like pathology. Moreover, compared with other IgG isotypes with undesired effector functions, mutant IgG4 Fc should prove useful in constructing novel therapeutic reagents to block immune molecule signalling in various diseases.

Acute allograft rejection has been recognized as a major impediment to improved success in renal transplantation. Timely detection and control of rejection are very important for the improvement in long-term renal allograft survival. Thus, biomarkers for early diagnosis of acute rejection are required urgently to clinical medication. This study seeks to search for such biomarker candidates by comparing patients' pre-treatment urinary protein profiling with their post-treatment urinary protein profiling. A total of 15 significantly and consistently down-regulated protein candidates were identified. Among them, alpha-1-antichymotrypsin precursor (AACT), tumor rejection antigen gp96 (GP96) and Zn-Alpha-2-Glycoprotein (ZAG) were selected for further analysis. The results indicated that Western Blot assay of AACT, GP96 and ZAG had advanced the diagnosis time of acute renal rejection by 3 days, compared with current standard clinical observation and laboratory examination. Furthermore, the double-blind detection revealed that the accuracy, sensitivity and specificity of the diagnosis of acute renal rejection of AACT, GP96 and ZAG were 66.67%/100%/60%, 83.33%/100%/80% and 66.67%/100%/60%, respectively, and 100%/100%/100% in combination. In conclusion, urinary protein AACT, GP96 and ZAG could be a set of potential biomarkers for early non-invasive diagnosis of the acute rejection after renal transplantation.

The precociously sexual maturation in large yellow crocker Pseudosciaena crocea has become a serious problem. In an attempt to solve this problem, the production of sterile triploids could be an effective strategy. In this study, triploid P. crocea was obtained by subjecting fertilized eggs to pressure shock. Flow-cytometry analysis was used to assess ploidy level. In terms of triploid rate and hatching rate, the optimal conditions of pressure shock for triploidy induction in P. crocea were 7500 psi for 3 min shock at 3 min after fertilization at 20 degrees C. With the application of these parameters, 100% triploid fish were produced. During the first rearing year, triploid P. crocea had a similar growth performance compared with its diploid counterpart before the age of 8 months and showed a significant advantage at the age of 10 and 12 months in body weight and body length (P < 0.05). At the age of 12 months, the carcass weight of triploids was markedly higher than that of diploid control, and gonadal somatic index was significantly lower than that of their diploid control. During the first rearing year, survival in triploid group was 76.44%, inferior to its diploid control (83.21%).

Cytochrome P450s metabolize the naturally occurring nephrotoxin aristolochic acid. Using liver-specific cytochrome P450 reductase-null mice we found that a low but lethal dose of aristolochic acid I was ineffective in wild-type mice. Induction of hepatic CYP1A by 3-methylcholanthrene pretreatment markedly increased the survival rate of wild type mice given higher doses and these mice were protected from aristolochic acid I-induced renal injury. Clearance of aristolochic acid I in null mice was slower compared to control and the 3-methylcholanthrene-pretreated wild type mice. The levels of aristolochic acid I in the kidney and liver were much higher in null mice but much lower in 3-methylcholanthrene-treated compared to control wild type mice. Hepatic microsomes from 3-methylcholanthrene-treated wild type mice had greater activity compared to untreated mice. Finally, aristolochic acid I was more cytotoxic than its major metabolite aristolactam I and this cytotoxicity was decreased in human renal tubular epithelial HK2 cells in the presence of a reconstituted hepatic microsome-cytosol (S9) system. These results indicate that hepatic P450s play an important role in metabolizing aristolochic acid I into less toxic metabolites and thus have a detoxification role in aristolochic acid I-induced kidney injury.

Raw corn starch (RCS), raw tapioca starch (RTS), raw potato starch (RPS), pre-gelatinized corn starch (PCS), pre-gelatinized tapioca starch (PTS) and pre-gelatinized potato starch (PPS) were evaluated as starch sources in diets for yellowfin seabream Sparus latus in a 56-day growth trial. Seven isonitrogenous semi-purified diets comprising a non-starch cellulose control diet and the six different starch sources holding 200 g kg−1 starch each were prepared and fed to triplicate groups of juvenile yellowfin seabream S. latus. Fish were fed for 8 weeks. Weight gain (WG) and specific growth rate (SGR) for fish fed RCS, RTS and RPS diets were equal, as well as for fish fed PCS, PTS and PPS diets, but values in groups fed the raw starch sources were significantly higher compared with fish fed the pre-gelatinized starches. Feed efficiency and protein efficiency ratio in fish fed different starch source diets showed no significant differences but were significantly higher than those fed a non-starch control diet. Protein productive value was improved by starch incorporation to diets. PCS, PTS or PPS groups showed lower feed intake compared with RCS, RTS or RPS groups, and the differences were significant between PCS, PPS and RCS, RPS groups. Whole-body protein and ash contents and muscle compositions were not affected by different starch sources. Whole-body and liver lipid contents, liver moisture and glycogen contents were significantly affected by starch source. Values of hepatosomatic index, intraperitoneal fat ratio, viscerosomatic index and condition factor did not vary between experimental treatments. Plasma total protein concentration for RCS, RTS or RPS fed fish was significantly higher than that for PCS, PTS or PPS fed fish, but significantly lower than that for non-starch fed fish. Plasma cholesterol and triacylglycerol concentrations were unaffected by starch source, but were significantly higher in fish fed the non-starch control diets. Plasma glucose concentrations in all dietary groups were relatively stable. In conclusion, raw corn, tapioca and potato starches at a 200 g kg−1 inclusion level were well utilized as energy sources by yellowfin seabream, which was evidenced by better WG and SGR. Pre-gelatinization of the starches had no positive effect on starch utilization.

Aim: Angiotensin II is believed to play an important role in tissue repair and remodeling in lungs by the angiotensin type I (AT1) receptor via a number of potential mechanisms. However, the role of the AT1 receptor in early lung injury has not been characterized. Methods: Bleomycin-induced pulmonary fibrosis (PF) in rats was utilized to value the treatment with valsartan, an AT1 receptor antagonist, by measurement of body weight, wet weight of the left lung, hydroxyproline content, mRNA expression of collagen I/III, and the degree of fibrosis in lung tissues on d 21. Tissue injury in the early phase was assessed on d 1, 3 and 7 by apoptosis, malondialdehyde content, myeloperoxidase activity, inflammatory cell count and protein content. Angiotensin converting enzyme (ACE) activity and the AT1 receptor in lung tissues were analyzed by biochemistry method and Western blotting, respectively. Results: Valsartan ameliorated PF induced by bleomycin in the rats on d 21. After bleomycin was injected intratracheally, increases in the lung AT1 receptor and ACE activity were observed by d 1, 3 and 7. Lung injury deteriorated in the early phase. Valsartan reduced the increase of the AT1 receptor, ACE activity and lung injury induced by bleomycin in the early phase. Conclusion: These observations suggest that angiotensin II may play a potent role in early lung injury via the AT1 receptor. AT1 receptor antagonists should be assessed as potential new therapies for fibrotic lung disease.

In this work, we developed a sensitive and universal aptasensor for nuclear factor kappa B (NF-κB) detection based on peroxidase-like mimic coupled DNA nanoladders for signal amplification. The dsDNA formed by capture DNA S1 and NF-κB binding aptamer (NBA) was firstly assembled on electrode surface. The presence of target NF-κB then led to the leave of NBA from electrode surface and thus provided the binding sites for immobilizing initiator to trigger in situ formation of DNA nanoladders on electrode surface. Since the peroxidase-like mimic manganese (III) meso-tetrakis (4-Nmethylpyridyl)-porphyrin (MnTMPyP) interacts with DNA nanoladders via groove binding, the insoluble benzo-4-chlorohexadienone (4-CD) precipitation derived from the oxidation of 4-chloro-1-naphthol (4-CN) could be formed on electrode surface in the presence of H2O2, resulting in a significantly amplified EIS signal output for quantitative target analysis. As a result, the developed aptasensor showed a low detection limit of 7pM and a wide linear range of 0.01-20nM. Featured with high sensitivity and label-free capability, the proposed sensing scheme can thus offer new opportunities for achieving sensitive, selective and stable detection of different types of target proteins.

Background: The immunoregulatory properties of mesenchymal stem cells (MSCs) have been well characterized in vitro. However, the role of MSCs in organ transplantation remains unclear. The purpose of this study was to examine the role of indoleamine 2, 3-dioxgenase (IDO)-transfected MSCs in immunoregulation both in vitro and in vivo. Methods: Wild-type (WT) MSCs, empty lentivirus-transfected MSCs (Lenti-MSCs) or IDO-lentivirus-transfected MSCs (IDO-MSCs) were cocultured with peripheral blood mononuclear cells (PBMCs) or CD4CD25 regulatory T (Treg) cells to examine the impact of IDO on the immunoregulatory properties of MSCs in vitro. WT-MSCs, Lenti-MSCs or IDO-MSCs (2 × 10/kg) were intravenously injected into rabbit renal transplant recipients immediately after surgery to examine the role of IDO-MSCs in tolerance induction in vivo. Results: Lentivirus infection of MSCs resulted in stable expression of IDO. The IDO-MSCs inhibited the proliferation of CD4CD25 effector T cells to a greater extent than WT-MSCs. Coculture of PBMCs and IDO-MSCs induced a higher percentage of CD4CD25Foxp3 Treg cells in PBMCs. Additionally, the antigen-specific suppressive function of these CD4CD25 Treg cells was increased. The IDO-MSCs-treated Treg cells showed upregulated expression of cytotoxic T-lymphocyte-associated antigen 4 and increased secretion of IL-10 and TGF-β. Low doses of IDO-MSCs prolonged graft survival and induced tolerance by inducing antigen-specific CD4CD25 Treg cells, as evidenced by the finding that IDO-MSCs-treated kidney transplant recipients accepted donor-specific skin grafts but rejected third-party grafts. Conclusions: The IDO increased the direct immunoregulatory properties of MSCs. The IDO-MSCs enhanced the expression and function of CD4CD25 Foxp3 Treg cells and induced allograft tolerance.

Background Advanced oxidation protein products (AOPPs) are new uremic toxins reported by Witko-Sarsat in 1996, which are associated with the pathogenesis of atherosclerosis. However, the mechanisms by which AOPPs enhance atherosclerosis have not been fully understood. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine which stimulates migration of monocytes and plays a critical role in the development of atherosclerosis. In this study, we investigated the effect of AOPPs on MCP-1 expression in cultured vascular smooth muscle cells (VSMCs). Methods VSMCs were cultured and then co-incubated with AOPP (200 micromol/L, 400 micromol/L) for different times with or without pretreatment with specific p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580. RT-PCR and Western blott were used to detect MCP-1 mRNA and protein expression at different time points after AOPP stimulation in rat smooth muscle cells. Western blot was used to detect the expression of phosphorylated p38 MAPK. Results Treatment of VSMC with AOPPs resulted in a significant increase of the expression of MCP-1 mRNA and protein in time- and dose-dependent manner, and could activated p38 MAPK. Pretreatment of VSMCs with SB203580 resulted in a dose-dependent inhibition of AOPPs-induced MCP-1 mRNA and protein expression. Conclusions AOPPs can stimulate MCP-1 expression via p38 MAPK in VSMCs. This suggests that AOPPs might contribute to the formation of atherosclerosis through this proinflammatory effect.

After comparing mortality and clinical signs in rats receiving different dexamethasone treatments, we investigated whether 0.5 mg/kg/d dexamethasone could delay pulmonary fibrosis induced by bleomycin and its time course (1, 3, 7, 14, 21 and 28 days). Tissue injury was assessed by apoptosis, lactate dehydrogenase (LDH) release, malondialdehyde content, and protein content; and inflammation was measured in terms of myeloperoxidase (MPO) activity, inflammatory cell count, and the mRNA expression of pro/inflammatory cytokines. Fibrogenic activity was analyzed by measuring the mRNA expression of fibrotic cytokines in tissue, and the promotion of fibroproliferation and synthesis of collagen type I by bronchoalveolar lavage fluids in vitro; and fibrosis was assessed by measuring the hydroxyproline content and collagen-I mRNA expression, and by histology. Bleomycin treatment induced tissue injury, inflammation and fibrogenic activity in lung, and led to fibrosis. Treatment with dexamethasone diminished the extent of fibrosis by strongly reducing inflammation, lung damage, and fibrogenic activity. These results demonstrate that the progression of bleomycin-induced pulmonary fibrosis in rats can be delayed by dexamethasone treatment, which appeared to alleviate not only inflammation but also lung damage and fibrogenic activity, indicating a possible new role for dexamethasone in the treatment of fibrosis.

BXSB mice spontaneously develop an autoimmune syndrome characterized by hypergammaglobulinemia, autoantibody production, and the development of fatal glomerulonephritis that closely resembles systemic lupus erythematosus (SLE) in humans. While blocking positive T cell co-stimulation has shown effectiveness in preventing the onset of murine lupus, deliberate delivering negative co-stimulation to halt unwanted T and B cell activation has not been tested. We developed a recombinant adenovirus containing the full-length mouse PD-L1 gene (Ad.PD-L1) to engage the immunoinhibitory receptor PD-1 on activated lymphocytes to prevent lupus nephritis in BXSB mice. This strategy was further reinforced by concomitant injection of anti-ICOSL(B7h) mAb to block ICOS-mediated co-stimulation. The combined therapy dramatically delayed the onset of proteinuria, effectively inhibited IgG autoantibody production, and significantly reduced hypercellularity and deposition of IgG in glomeruli, resulting in almost complete amelioration of lupus nephritis in these animals. Our results indicate the therapeutic potential of simultaneous stimulation of PD-1-mediated pathway and blockade of ICOS-B7h co-stimulation in the prevention of human lupus nephritis.

The current therapeutic approaches for pulmonary fibrosis, which is characterized by fibroblast proliferation and extracellular matrix remodeling, are unsatisfactory. Feitai, consisting of several herbs, is a folk formula for pulmonary tuberculosis therapy in China. To investigate the effects of Feitai on pulmonary fibrosis, Feitai was administered orally to bleomycin (BLM)-treated rats, and the lung toxicity effects were evaluated according to inflammatory cell count, protein concentration, and lactate dehydrogenase (LDH) activity in the bronchoalveolar lavage fluid (BALF), malondialdehyde level and hydroxyproline content in lung tissue 28 days post-BLM. Serial sections of the lung were stained with hematoxylin and eosin (HE) and Masson trichrome, respectively. The degree of fibrosis was assessed quantitatively using LEICA QWin image analyzer. Results showed that Feitai inhibited BLM-induced lung fibrotic lesions in a dose-dependent manner as reflected by decreased the lung hydroxyproline content and lung fibrosis fraction 28 days after BLM instillation. Treatment with Feitai also significantly ameliorated the BLM-induced lung toxicity effects detected in BALF and lung tissue. The effects in vitro on WI-38 human lung fibroblast cell line showed that Feitai significantly reduced the cell proliferation and transforming growth factor (TGF)–β stimulated type I collagen synthesis. These results strongly demonstrate that Feitai may be useful in the treatment of pulmonary fibrosis.

Pulmonary fibrosis is a common consequence of numerous pulmonary diseases. The current therapeutic approaches for this condition are unsatisfactory. Feitai, a composite formula consisting of several herbs, is used in China as a folk remedy for treating patients with pulmonary tuberculosis. In this study, we extensively investigate the effects and mechanisms of Feitai on bleomycin (BLM)-induced pulmonary fibrosis in rats. One hundred and twenty male Sprague-Dawley rats were randomly divided into four groups, referred to as the saline-water, saline-Feitai, BLM-water, and BLM-Feitai groups. Following a single instillation of BLM (5 mg/kg) or saline, rats were orally administered Feitai at a dose of 3 g/kg body weight or sterilized distilled water once daily. Rats were killed at 7, 14, or 28 d post-BLM. Inflammatory cell count, protein concentration, and lactate dehydrogenase activity in bronchoalveolar lavage fluid were measured, and myeloperoxidase activity and lipid peroxide content in lung homogenates were analyzed. Treatment with Feitai inhibited lung fibrotic progression induced by BLM, as indicated by the decrease in lung hydroproline content and lung fibrosis score at 28 d post-BLM. This was accompanied by significant amelioration of BLM-induced body weight loss, lung edema, and inflammatory response during the development of lung injury in the acute phase. The results strongly indicate the beneficial effects of Feitai in protecting against BLM-induced pulmonary fibrosis. Furthermore, the inflammatory response and lipid peroxidation were inhibited by Feitai, suggesting that the effect of this formula on BLM-induced lung injury and fibrosis is associated with antiinflammatory and antioxidant properties.

Background/Aims: To reveal the potential pathogenesis of ischemia/reperfusion (I/R) injury. Methods: GSE9943 were downloaded from Genome Expression Omnibus database, including I/R and control samples for both Brown Norway (BN) and Sprague Dawley (SD) rats (3 rats/each group). Then differentially expressed genes (DEGs) were identified by limma package. miRNA-target gene network pairs were predicted using WebGestalt, and protein-protein interactions (PPI) were identified based on STRING database, followed by the networks construction using Cytoscape. Next, ClusterONE was used for modules screening. Furthermore, functional analyses were performed to common DEGs and genes. Results: Totally, 23 common DEGs of BR and SD rats were screened, enriched in functions, such as regulation of cellular protein metabolic process, response to wounding, proteinaceous extracellular matrix, and Enzyme inhibitor activity. MIR-29A, MIR-29B and MIR-29C were discovered both in up- and down-regulated miRNA-target gene networks. Genes in the PPI network were significantly disturbed in p53 signaling, complement and coagulation cascades pathway. Four modules were found significantly disturbed cytochrome P450, Serine/threonine protein kinase, calcium binding and Transient receptor potential channel protein domains. Conclusion: During I/R injury, many genes mutated, interrupting several biological functions, pathways and protein domains. MIR-29C and TRPC6 were suggested to be potential novel targets for this disease.

BACKGROUND Hyperphosphataemia in patients with advanced chronic kidney disease (CKD) is associated with adverse outcomes, including vascular calcification and higher mortality rates. While phosphate lowering is an integral aspect of CKD management, the efficacy and safety of phosphate binders in a contemporary cohort of Chinese haemodialysis patients (who have different genetics and dietary patterns than other populations) has not been previously described. Moreover, sparse data are available on strategies for optimal dose titration when transitioning from a calcium-based to a polymer-based phosphate binder. METHODS This randomized, double-blind, dose-titration study compared sevelamer carbonate (starting dose 800 mg three times daily) with placebo over 8 weeks' duration in Chinese CKD patients on haemodialysis. Patients were required to be using calcium-based binders prior to study start. RESULTS In all, 205 patients were randomized (sevelamer, n = 135; placebo, n = 70); mean age was 48.6 years, 61% were male and the mean time on dialysis was 4.4 years. The mean serum phosphorus decreased significantly in patients treated with sevelamer carbonate [change -0.69 ± 0.64 mmol/L (-2.14 ± 1.98 mg/dL)] but remained persistently elevated with placebo [change -0.06 ± 0.57 mmol/L (-0.19 ± 1.76 mg/dL)] (P < 0.0001). When compared with placebo, sevelamer carbonate treatment resulted in statistically significant greater mean reductions from baseline in serum total (-17.1 versus -3.3%) and low-density lipoprotein cholesterol (-33.5 versus-7.6%) (P < 0.0001 for both). Sevelamer carbonate was well tolerated with 96% adherence compared with 97% adherence in the placebo arm. Overall, adverse events experienced by patients in the sevelamer carbonate and placebo treatment groups were similar and consistent with their underlying renal disease. CONCLUSIONS This study demonstrated that hyperphosphataemia developed quickly following the cessation of phosphate binders and remained persistently elevated in end-stage CKD in the placebo-treated group. Gradually titrating up sevelamer carbonate from an initial dose of 2.4 g/day to an average daily dose of 7.1 ± 2.5 g/day was well tolerated, safe and efficacious in contemporary Chinese haemodialysis patients.

BackgroundAbelmoschus manihot, a single medicament of traditional Chinese medicine, has been widely used to treat kidney disease. This is the first randomized controlled clinical trial to assess its efficacy and safety in patients with primary glomerular disease.Study DesignProspective, open-label, multicenter, randomized, controlled, clinical trial.Setting & ParticipantsFrom May 2010 to October 2011, a total of 417 patients with biopsy-proven primary glomerular disease from 26 hospitals participated in the study.InterventionsA manihot in the form of a huangkui capsule, 2.5g, 3 times per day; losartan potassium, 50mg/d; or combined treatment, a huangkui capsule at 2.5g 3 times per day, was combined with losartan potassium, 50mg/d. The duration of intervention was 24 weeks.Outcomes & MeasurementsThe primary outcome was change in 24-hour proteinuria from baseline after treatment. Change in estimated glomerular filtration rate (eGFR) from baseline after treatment was a secondary outcome. The 24-hour proteinuria was measured every 4 weeks and eGFR was measured at 0, 4, 12, and 24 weeks.ResultsMean baseline urine protein excretion was 1,045, 1,084, and 1,073mg/d in the A manihot, losartan, and combined groups, respectively, and mean eGFR was 108, 106, and 106mL/min/1.73m2, respectively. After 24 weeks of treatment, mean changes in proteinuria were protein excretion of −508, −376, and −545mg/d, respectively (P=0.003 for A manihot vs losartan and P0.05), and there were no severe adverse events in any group.LimitationsResults cannot be generalized to those with nephrotic syndrome or reduced eGFR.ConclusionsA manihot is a promising therapy for patients with primary kidney disease (chronic kidney disease stages 1-2) with moderate proteinuria.

Background: Prulifloxacin is a promising fluoroquinolone antibiotic. A multicentre, double-blind, randomized clinical study was designed to evaluate its efficacy and safety compared to that of levofloxacin for the treatment of respiratory and urinary infections of Chinese patients. Methods: A total of 267 patients were enrolled and each was randomly assigned to either the treatment or the control group. Prulifloxacin 264.2 mg (equivalent to ulifloxacin 200 mg) b.i.d. or levofloxacin hydrochloride 200 mg b.i.d. was administered orally for 5–14 days according to a patient’s condition. The clinical response, bacterial eradication and incidence of adverse events were evaluated. Results: Two hundred and forty-three patients completed the study. For the modified intention-to-treat population, the cure and effective rates were 45.53 and 82.93% in the prulifloxacin group and 49.18 and 83.61% in the levofloxacin group. For the per-protocol analysis population, the cure and effective rates were 45.90 and 83.61% in the prulifloxacin group and 49.59 and 83.47% in the levofloxacin group. The bacterial eradication rates were 96.59 and 95.35%, and the drug-related adverse event rates were 7.87 and 5.51% in the prulifloxacin and levofloxacin group, respectively. The cure rate and efficacy rate of respiratory and urinary tract infections of the levofloxacin group were better than those of the prulifloxacin group. However, the difference between the 2 groups was not statistically significant (p > 0.05). Conclusion: Prulifloxacin is as effective and well tolerated as levofloxacin in the treatment of respiratory and urinary tract infections.

CD4 + CD25 + regulatory T cells (Tregs) have recently been the subject of intense research due to their strong immunosuppressive effect. Increasing evidence suggests that IL-15 plays an important role in Tregs biology. Nevertheless, the mechanism by which IL-15 performs this function remains to be fully elucidated. To address this question, we isolated Tregs from human peripheral blood, and utilized IL-15, dendritic cells (DCs), or DCs combined with IL-15, to examine the proliferation of Tregs and to explore related molecular mechanisms. Here, we show that IL-15 can induce the proliferation of Tregs in the presence of DCs. The induction is mediated by DCs presenting IL-15 in trans to Tregs. Simultaneously, DCs-derived IL-2, regulated by IL-15, may also play a supportive role. After IL-15 withdrawal, IL-15 trans-endosomal recycling in DCs contributes to the proliferation of Tregs. The activation of Akt, Erk1/2 and STAT 5 , and the degradation of p27 kip1 may be involved in this process. These findings might explain the proliferation of Tregs in the absence of IL-2 in vivo and provide a novel method to achieve large-scale proliferation of Tregs in vitro in order to obtain cell numbers sufficient for immunotherapy.

Emodin, an anthraquinone derived from a plant and fungi, has been reported to possess potential genotoxicity, but the mechanism is not entirely clear. Here, we report that emodin causes DNA double-strand breaks (DSBs) through stabilization of topoisomerase (Topo) II-DNA cleavage complexes and inhibition of ATP hydrolysis. In our study, emodin did not induce mutagenecity in the salmonella mutation assay but caused genotoxicity in the thymidine kinase gene mutation assay and in the micronucleus test. Moreover, emodin induced DNA DSBs demonstrated by induction of comet tails, the expression of phosphorylated histone H2AX, and phosphorylation of ataxia telangiectasia mutated. Our studies also revealed that emodin exerted strong inhibitory activity against Topo II in the supercoiled pBR322 relaxation assay and in Topo II-mediated kinetoplast DNA decatenation, similar to the previous report. We also showed that the inhibitory effect of emodin on Topo II was because of its ability to stabilize Topo II-DNA complexes and to inhibit the ATP hydrolysis of Topo II. Furthermore, emodin was found to trigger DNA DSBs in a Topo II-dependent manner using the Topo II catalytic inhibitor aclarubicin and in Topo II-deficient mitoxantrone-resistant variant HL-60/MX2 cells. Together, these results suggest that in emodin-induced DNA DSBs and genotoxicity, stabilization of Topo II-DNA cleavage complexes and inhibition of ATP hydrolysis play an important role.

Ethnopharmacological relevance Longdan Xieganwan, which contains Aristolochia species, is a traditional Chinese prescription. It has been used for thousands of years to “enhance liver”. However, many cases of Longdan Xieganwan induced nephropathy were reported recently. Aim of the study This study was designed to compare the possible toxic effects of Longdan Xieganwan and three different Aristolochia species, i.e. Akebia trifoliate (Thunb.) koid (Akebia trifoliate), Akebia quinata (Thunb.) Decne. (Akebia quinata) and Caulis aristolochiae manshuriensis (Aristolochia manshuriensis). Materials and methods Mice were orally administered these drugs for 28 days. Clinical signs, body weights, serum biochemistry, organ weights and histopathology were examined. Results Significantly decreased body weights and obvious nephropathy were noticed in the Aristolochia manshuriensis groups at doses higher than 0.24 g/kg/d. A few endothelial cell degenerations in renal glomerulus were observed in the Akebia trifoliate group at a high-dose of 2.00 g/kg/d. No significant changes were observed in the other groups. Conclusions The no-observed-adverse-effect levels (NOAELs) for Aristolochia manshuriensis, Akebia trifoliate, Akebia quinata and Longdan Xieganwan in this study for mice were 0.06 g/kg/d, 0.40 g/kg/d, higher than 3.00 g/kg/d and higher than 10.00 g/kg/d, which were equivalent to 0.25 times, 5 times, 25 times and 10 times of normal human dose in clinical prescription, respectively.

Toosendanin (Tsn), a triterpenoid extracted from Melia toosendan Sieb et Zucc, possesses different pharmacological effects in human and important values in agriculture. However, liver injury has been reported when toosendanin or Melia-family plants, which contain toosendanin are applied. The mechanism by which toosendanin induces liver injury remains largely unknown. Here we reported that toosendanin induced primary rat hepatocytes death by mitochondrial dysfunction and caspase activation. Toosendanin led to decrease of mitochondrial membrane potential, fall in intracellular ATP level, release of cytochrome c to cytoplasm, activation of caspase-8, 9, and 3 and ultimately cell death. Level of reactive oxygen species (ROS) was also increased in hepatocytes after incubation with toosendanin. Catalase, the H2O2-decomposing enzyme, can prevent the reduction in ATP level and protect hepatocytes from toosendanin-induced death. The ERK1/2 (p44/42 MAP kinases) and JNK (c-Jun N-terminal kinase) were activated, but p38 MAPK was not activated by toosendanin. Inhibition of ERK1/2 activation sensitized hepatocytes to death and increased activity of caspase-9 and 3 in response to toosendanin. Inhibition of JNK attenuated toosendanin-induced cell death. These results suggested that toosendanin causes death of primary rat hepatocytes by mitochondrial dysfunction and caspase activation. Generation of ROS and MAP kinases activation might be involved in this process.

Aristolochic acid (AA), a natural nephrotoxin and carcinogen, can induce a progressive tubulointerstitial nephropathy. However, the mechanism by which AA causes renal injury remains largely unknown. Here we reported that the mitochondrial permeability transition (MPT) plays an important role in the renal injury induced by aristolochic acid I (AAI). We found that in the presence of Ca(2+), AAI caused mitochondrial swelling, leakage of Ca(2+), membrane depolarization, and release of cytochrome c in isolated kidney mitochondria. These alterations were suppressed by cyclosporin A (CsA), an agent known to inhibit MPT. Culture of HK-2 cell, a human renal tubular epithelial cell line for 24 h with AAI caused a decrease in cellular ATP, mitochondrial membrane depolarization, cytochrome c release, and increase of caspase 3 activity. These toxic effects of AAI were attenuated by CsA and bongkrekic acid (BA), another specific MPT inhibitor. Furthermore, AAI greatly inhibited the activity of mitochondrial adenine nucleotide translocator (ANT) in isolated mitochondria. We suggested that ANT may mediate, at least in part, the AAI-induced MPT. Taken together, these results suggested that MPT plays a critical role in the pathogenesis of HK-2 cell injury induced by AAI and implied that MPT might contribute to human nephrotoxicity of aristolochic acid.