Postbiotic of Pediococcus acidilactici GQ01, a Novel Probiotic Strain Isolated from Natural Fermented Wolfberry, Attenuates Hyperuricaemia in Mice through Modulating Uric Acid Metabolism and Gut Microbiota.

Hyperuricaemia (HUA) is a disorder of purine metabolism, which manifests itself as an increase in uric acid production and a decrease in uric acid excretion, as well as a change in the structure of the intestinal microbiota. Most of the drugs currently used to treat HUA have significant side effects, and it is essential to find a treatment for HUA that is free of side effects. In this study, a novel strain, Pediococcus acidilactici GQ01, was screened from natural fermented wolfberry. The effects of both live bacteria GQ01 and its heat-killed G1PB postbiotic on HUA were investigated. The results showed that both probiotic GQ01 and G1PB postbiotics could effectively decrease blood uric acid, creatinine, and urea nitrogen levels in the HUA mice model. P. acidilactici GQ01 was more effective in inhibiting ADA activity, while G1PB postbiotics was more effective in inhibiting XOD activity. Meanwhile, GQ01 and G1PB were able to ameliorate liver and kidney tissue injury, upregulate the expression of ABCG2 in kidney and XOD gene in liver, downregulate the protein expression of URAT1 and GLUT9 in kidney, and therefore reduce the value of blood uric acid by decreasing the uric acid reabsorption and increasing the excretion of uric acid. Additionally, both probiotics and postbiotics could regulate the intestinal microbiota structure of HUA mice, so as to bring the dysfunctional intestinal composition back to normal. Furthermore, P. acidilactici GQ01 and G1PB postbiotics can increase the levels of acetic acid, propionic acid, and butyric acid in the intestinal tract, improve the intestinal function, and maintain the healthy homeostatic state of the intestinal tract. In summary, P. acidilactici GQ01 and its G1PB postbiotics may be developed as functional food or drug materials capable of treating HUA.

Research review on methods of grassland health assessment.

Grassland health refers to the degree to which the integrity of soil and ecological processes is maintained, which primarily reflects the health status and productivity of grasslands. Evaluating the degree of grassland health is vital for the sustainable develop of grasslands. There are many methods for evaluating grassland health, with advantages and disadvantages for each one. However, there is still a lack of systematic literature offering an overview of methods of grassland health assessment and their applicability. We summarized 10 methods of grassland health assessment, including vigor-organization-resilience (VOR) index evaluation model, condition-vigor-organization-resilience (CVOR) index evaluation model, principal component analysis method, analytic hierarchy process, cluster analysis method, grey relational analysis, pressure-state-response evaluation model, fuzzy comprehensive evaluation method, comprehensive evaluation model of grassland health, and evaluation model using remote sensing technology. The advantages, disadvantages, and applicability of these methods were discussed, aiming to provide scientific basis for selecting more suitable methods of grassland health assessment for different scenarios in the future.

Novel insights into aerobic duration control-based partial nitritation in source-separated blackwater treatment: Growth type, inoculation source, and comammox threat.

Aerobic duration control (ADC), whereby aeration is terminated before nitrite is extremely oxidized during the nitrification process, is an effective strategy to achieve partial nitritation (PN) for blackwater. This study evaluated the effects of microbial growth type, influent ammonia-oxidizing organisms (AOO), and comammox bacteria from seeding sludge to ADC-based PN. The long-term operation of lab-scale reactors and model simulations were implemented to select the best growth type. The biofilm formed on the inner wall of the activated sludge reactor decreased the nitrite accumulation ratio (NAR) from 99.2% to 77.2%. Meanwhile, the NAR of the pure-biofilm reactor decreased from 95.9% to 47.8%. The deteriorated PN of the biofilm-related reactors was due to the extended solid retention time and increased substrate saturation constants of AOOs compared with those of nitrite-oxidizing organisms (NOO). Periodic biofilm carrier regeneration and biofilm thickness control can recover PN performance but are difficult to implement. In contrast, the optimized activated sludge reactor exhibited high (NAR >94%) and stable (>3 months) PN performance when treating real blackwater. Nitrifiers were found in blackwater, and chemically enhanced high-rate activated sludge pretreatment removed more NOOs than AOOs (41.8% vs. 24.3%) and increased the influent AOO/NOO ratio. Interestingly, the influent AOOs supported fast PN start-up in the moving-bed biofilm reactor without the initial inoculation of activated sludge. Moreover, model simulations verified that high and stable PN could also be realized in an activated sludge reactor by the continuous inoculation of influent AOOs, which is a novel PN start-up strategy. Metagenomic analyses showed that the comammox bacteria from the seeding sludge eventually disappeared owing to their intrinsic specific growth rates and free ammonia inhibition. The findings of this study will provide insightful guidelines for PN application in decentralized and semi-centralized wastewater treatment systems.

Reduced electric field and gas temperature effects on chemical product dynamics in air surface dielectric barrier discharges: from macro-physical parameters to micro-chemical mechanisms.

To gain insights into the mechanisms of plasma chemical product interactions, the dynamic changes of the surface dielectric barrier discharge (SDBD) products are experimentally related to the reduced electric field and gas temperature. The higher applied voltage and frequency cause faster product changes from the O3-containing to the O3-free state, while raising the electron energy and gas temperature. The electron energy affects the electron collision reactions and the production of various reactive species, steering the chemical reactions towards the predominant production of NO over O3. The gas temperature affects the generation and quenching rates of the key products. Collectively, this work bridges macro-physical parameters and micro-chemical mechanisms through the electron energy and gas temperature effects, and contributes to better understanding of the physico-chemical processes in low-temperature plasmas.

Combined Effect of CYP2C19 Genetic Polymorphisms and C-Reactive Protein on Voriconazole Exposure and Dosing in Immunocompromised Children.

Background: Pediatric patients have significant interindividual variability in voriconazole exposure. The aim of the study was to identify factors associated with voriconazole concentrations and dose requirements to achieve therapeutic concentrations in pediatric patients. Methods: Medical records of pediatric patients were retrospectively reviewed. Covariates associated with voriconazole plasma concentrations and dose requirements were adjusted by using generalized linear mixed-effect models. Results: A total of 682 voriconazole steady-state trough concentrations from 91 Chinese pediatric patients were included. Voriconazole exposure was lower in the CYP2C19 normal metabolizer (NM) group compared with the intermediate metabolizer (IM) group and the poor metabolizer (PM) group (p = 0.0016, p < 0.0001). The median daily dose of voriconazole required to achieve therapeutic range demonstrated a significant phenotypic dose effect: 20.8 mg/kg (range, 16.2-26.8 mg/kg) for the CYP2C19 NM group, 18.2 mg/kg (range, 13.3-21.8 mg/kg) for the CYP2C19 IM group, and 15.2 mg/kg (range, 10.7-19.1 mg/kg) for the CYP2C19 PM group, respectively. The extent of impact of C-reactive protein (CRP) levels on voriconazole trough concentrations and dose requirements varied between CYP2C19 phenotypes. Increases of 20, 120, 245, and 395 mg/L from 5 mg/L in CRP levels were associated with increases in voriconazole trough concentration by 22.22, 50, 64.81, and 75% respectively, in the NM group; by 39.26, 94.48, 123.93, and 146.63%, respectively, in the IM group; and by 17.17, 37.34, 46.78, and 53.65%, respectively, in the PM group. Meanwhile, increases of 20, 120, 245, and 395 mg/L from 5 mg/L in CRP levels were associated with increases in voriconazole dose requirements by 7.15, 14.23, 17.35, and 19.43%, respectively, in the PM group; with decreases in voriconazole dose requirements by 3.71, 7.38, 8.97, and 10.03%, respectively, in the NM group; and with decreases by 4, 9.10, 11.05, and 12.35%, respectively, in the IM group. In addition, age and presence of immunosuppressants had significant effects on voriconazole exposure. Conclusions: Our study suggests that CYP2C19 phenotypes, CRP concentrations, age, and the presence of immunosuppressants were factors associated with the pharmacokinetic changes in voriconazole. There was heterogeneity in the effect of CRP on voriconazole plasma concentrations across different CYP2C19 genotypes. Combining relevant factors with dose adaptation strategies in therapeutic drug monitoring may help to reduce the incidence of subtherapeutic and supratherapeutic concentrations in clinical practice.

Predicting Blood Concentration of Tacrolimus in Patients With Autoimmune Diseases Using Machine Learning Techniques Based on Real-World Evidence.

Tacrolimus is a widely used immunosuppressive drug in patients with autoimmune diseases. It has a narrow therapeutic window, thus requiring therapeutic drug monitoring (TDM) to guide the clinical regimen. This study included 193 cases of tacrolimus TDM data in patients with autoimmune diseases at Southern Medical University Nanfang Hospital from June 7, 2018, to December 31, 2020. The study identified nine important variables for tacrolimus concentration using sequential forward selection, including height, tacrolimus daily dose, other immunosuppressants, low-density lipoprotein cholesterol, mean corpuscular volume, mean corpuscular hemoglobin, white blood cell count, direct bilirubin, and hematocrit. The prediction abilities of 14 models based on regression analysis or machine learning algorithms were compared. Ultimately, a prediction model of tacrolimus concentration was established through eXtreme Gradient Boosting (XGBoost) algorithm with the best predictive ability (R 2 = 0.54, mean absolute error = 0.25, and root mean square error = 0.33). Then, SHapley Additive exPlanations was used to visually interpret the variable's impacts on tacrolimus concentration. In conclusion, the XGBoost model for predicting blood concentration of tacrolimus on the basis of real-world evidence has good predictive performance, providing guidance for the adjustment of regimen in clinical practice.

Effector Avr4 in Phytophthora infestans Escapes Host Immunity Mainly Through Early Termination.

Effector genes play critical roles in the antagonistic interactions between plants and pathogens. However, knowledge of mutation mechanisms and evolutionary processes in effector genes and the contribution of climatic factors to the evolution of effector genes are fragmented but important in sustainable management of plant diseases and securing food supply under changing climates. Here, we used a population genetic approach to explore the evolution of the Avr4 gene in Phytophthora infestans, the causal agent of potato blight. We found that the Avr4 gene exhibited a high genetic diversity generated by point mutation and sequence deletion. Frameshifts caused by a single base-pair deletion at the 194th nucleotide position generate two stop codons, truncating almost the entire C-terminal, which is important for effector function and R4 recognition in all sequences. The effector is under natural selection for adaptation supported by comparative analyses of population differentiation (FST ) and isolation-by-distance between Avr4 sequences and simple sequence repeat marker loci. Furthermore, we found that local air temperature was positively associated with pairwise FST in the Avr4 sequences. These results suggest that the evolution of the effector gene is influenced by local air temperature, and the C-terminal truncation is one of the main mutation mechanisms in the P. infestans effector gene to circumvent the immune response of potato plants. The implication of these results to agricultural and natural sustainability in future climate conditions is discussed.

Evidence for a synergistic effect of post-translational modifications and genomic composition of eEF-1α on the adaptation of Phytophthora infestans.

Genetic variation plays a fundamental role in pathogen's adaptation to environmental stresses. Pathogens with low genetic variation tend to survive and proliferate more poorly due to their lack of genotypic/phenotypic polymorphisms in responding to fluctuating environments. Evolutionary theory hypothesizes that the adaptive disadvantage of genes with low genomic variation can be compensated for structural diversity of proteins through post-translation modification (PTM) but this theory is rarely tested experimentally and its implication to sustainable disease management is hardly discussed. In this study, we analyzed nucleotide characteristics of eukaryotic translation elongation factor-1α (eEF-lα) gene from 165 Phytophthora infestans isolates and the physical and chemical properties of its derived proteins. We found a low sequence variation of eEF-lα protein, possibly attributable to purifying selection and a lack of intra-genic recombination rather than reduced mutation. In the only two isoforms detected by the study, the major one accounted for >95% of the pathogen collection and displayed a significantly higher fitness than the minor one. High lysine representation enhances the opportunity of the eEF-1α protein to be methylated and the absence of disulfide bonds is consistent with the structural prediction showing that many disordered regions are existed in the protein. Methylation, structural disordering, and possibly other PTMs ensure the ability of the protein to modify its functions during biological, cellular and biochemical processes, and compensate for its adaptive disadvantage caused by sequence conservation. Our results indicate that PTMs may function synergistically with nucleotide codes to regulate the adaptive landscape of eEF-1α, possibly as well as other housekeeping genes, in P. infestans. Compensatory evolution between pre- and post-translational phase in eEF-1α could enable pathogens quickly adapting to disease management strategies while efficiently maintaining critical roles of the protein playing in biological, cellular, and biochemical activities. Implications of these results to sustainable plant disease management are discussed.

Altitudinal Heterogeneity of UV Adaptation in Phytophthorainfestans Is Associated with the Spatial Distribution of a DNA Repair Gene.

Climate change is considered a major threat to society and nature. UV irradiation is the most important environmental genotoxic agent. Thus, how elevated UV irradiation may influence human health and ecosystems has generated wide concern in the scientific community, as well as with policy makers and the public in general. In this study, we investigated patterns and mechanisms of UV adaptation in natural ecosystems by studying a gene-specific variation in the potato late blight pathogen, Phytophthora infestans. We compared the sequence characteristics of radiation sensitive 23 (RAD23), a gene involved in the nucleotide excision repair (NER) pathway and UV tolerance, in P. infestans isolates sampled from various altitudes. We found that lower genetic variation in the RAD23 gene was caused by natural selection. The hypothesis that UV irradiation drives this selection was supported by strong correlations between the genomic characteristics and altitudinal origin (historic UV irradiation) of the RAD23 sequences with UV tolerance of the P. infestans isolates. These results indicate that the RAD23 gene plays an important role in the adaptation of P. infestans to UV stress. We also found that different climatic factors could work synergistically to determine the evolutionary adaptation of species, making the influence of climate change on ecological functions and resilience more difficult to predict. Future attention should aim at understanding the collective impact generated by simultaneous change in several climate factors on species adaptation and ecological sustainability, using state of the art technologies such as experimental evolution, genome-wide scanning, and proteomics.

The Phytophthora infestans AVR2 Effector Escapes R2 Recognition Through Effector Disordering.

Intrinsic disorder is a common structural characteristic of proteins and a central player in the biochemical processes of species. However, the role of intrinsic disorder in the evolution of plant-pathogen interactions is rarely investigated. Here, we explored the role of intrinsic disorder in the development of the pathogenicity in the RXLR AVR2 effector of Phytophthora infestans. We found AVR2 exhibited high nucleotide diversity generated by point mutation, early-termination, altered start codon, deletion/insertion, and intragenic recombination and is predicted to be an intrinsically disordered protein. AVR2 amino acid sequences conferring a virulent phenotype had a higher disorder tendency in both the N- and C-terminal regions compared with sequences conferring an avirulent phenotype. In addition, we also found virulent AVR2 mutants gained one or two short linear interaction motifs, the critical components of disordered proteins required for protein-protein interactions. Furthermore, virulent AVR2 mutants were predicted to be unstable and have a short protein half-life. Taken together, these results support the notion that intrinsic disorder is important for the effector function of pathogens and demonstrate that SLiM-mediated protein-protein interaction in the C-terminal effector domain might contribute greatly to the evasion of resistance-protein detection in P. infestans.

The effectiveness of phenobarbital in patients with refractory status epilepticus undergoing therapeutic plasma exchange.

This study aimed to elucidate the therapeutic concentration range of phenobarbital (PB) for adults, as well as the influence of therapeutic plasma exchange (TPE) on plasma concentration of PB. We retrospectively reviewed consecutive patients diagnosed with refractory status epileptic (RSE) and treated with a bolus injection of PB as well as TPE, admitted to our neurocritical care unit from November 2015 to October 2016. Continuous electroencephalographic monitoring was performed routinely for these patients. TPE was performed using a continuous-flow cell separator. PB concentrations in the plasma and cerebrospinal fluid were measured by gas chromatography-mass spectrometer analysis before and after TPE. A total of seven patients were included; among these, one patient had RSE related to anti-N-methyl-D-aspartate receptor encephalitis, another patient had Hashimoto encephalopathy, and five patients had undetermined etiology. For patients with clinical and electrographic control (n=6), the plasma concentration of PB ranged from 138 to 243.7 µg/ml. In addition, of six paired plasma samples (before and after TPE) obtained from three patients, no significant differences between the concentrations of PB were detected (P=0.6), suggesting that TPE may not significantly affect the plasma concentration of PB. This study confirmed that PB more than 100 µg/ml was effective for adults with RSE. Moreover, TPE may not have an influence on the plasma concentration of PB.Video abstract: http://links.lww.com/WNR/A489.

Protective effects of Paeoniflorin against AOPP-induced oxidative injury in HUVECs by blocking the ROS-HIF-1α/VEGF pathway.

BACKGROUND: Paeoniflorin, a monoterpene glycoside, exerts protective vascular effects, showing good antioxidant properties. However, whether Paeoniflorin has protective effect against the oxidative damage induced by advanced oxidation protein products (AOPPs) in Human umbilical vein endothelial cells (HUVECs) is unknown, as is the underlying mechanism. PURPOSE: The present study was designed to investigate the effect of Paeoniflorin on oxidative damage of HUVECs and elucidate its underlying molecular mechanisms. METHODS: The fluorescence intensity of 2', 7'-dichlorofluorescein-diacetate (DCFH-DA) staining was detected for intracellular reactive oxygen species (ROS) production. The increases mitochondrial membrane potential (MMP) was measured via flow cytometry and confocal microscopy using MitoTracker® Deep Red/ MitoTracker® Green staining. The intracellular adenosine triphosphate (ATP) was measured by ATP Determination Kit according to the manufacturer's protocol. Nox2, Nox4, hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and nuclear factor-κB (NF-κB) p65 expressions were detected by western blot. RESULTS: Our results showed that Paeoniflorin increases MMP and ATP levels of HUVECs induced by AOPPs, and attenuates NF-κB p65 expression on HUVECs might mainly result from its antioxidant capability by suppressing ROS production. Moreover, we also found that Paeoniflorin can suppress HIF-1α and VEGF protein expression through a decrease of ROS production via down-regulation of Nox2/Nox4 expression in HUVECs. AOPP-induced RAGE mRNA up-regulation was blocked by Paeoniflorin treatment in HUVECs. CONCLUSION: Our results provided the first experimental that Paeoniflorin protects against AOPP-induced oxidative damage in HUVECs, mainly through a mechanism involving a decrease in ROS production by the inhibition of Nox2/Nox4 and RAGE expression; restored ATP depletion and mitochondria dysfunction via ROS suppression; and down-regulated HIF-1α/VEGF, possibly via the ROS-NF-κB axis.

Mechanism and kinetics of halogenated compound removal by metallic iron: Transport in solution, diffusion and reduction within corrosion films.

A detailed kinetic model comprised of mass transport (ktra), pore diffusion (kdif), adsorption and reduction reaction (krea), was developed to quantitatively evaluate the effect of corrosion films on the removal rate (kobs) of halogenated compounds by metallic iron. Different corrosion conditions were controlled by adjusting the iron aging time (0 or 1 yr) and dissolved oxygen concentration (0-7.09 mg/L DO). The kobs values for bromate, mono-, di- and tri-chloroacetic acids (BrO3-, MCAA, DCAA and TCAA) were 0.41-7.06, 0-0.16, 0.01-0.53, 0.10-0.73 h-1, with ktra values at 13.32, 12.12, 11.04 and 10.20 h-1, kdif values at 0.42-5.82, 0.36-5.04, 0.30-4.50, 0.30-3.90 h-1, and krea values at 14.94-421.18, 0-0.19, 0.01-1.30, 0.10-3.98 h-1, respectively. The variation of kobs value with reaction conditions depended on the reactant species, while those of ktra, kdif and krea values were irrelevant to the species. The effects of corrosion films on kdif and krea values were responsible for the variation of kobs value for halogenated compounds. For a mass-transfer-limited halogenated compound such as BrO3-, an often-neglected kdif value primarily determined its kobs value when pore diffusion was the rate-limiting step of its removal. In addition, the value of kdif might influence product composition during a consecutive dechlorination, such as for TCAA and DCAA. For a reaction-controlled compound such as MCAA, an increased krea value was achieved under low oxic conditions, which was favorable to improve its kobs value. The proposed model has a potential in predicting the removal rate of halogenated compounds by metallic iron under various conditions.

Effects of metal ions on disinfection byproduct formation during chlorination of natural organic matter and surrogates.

The effects of calcium, cupric, ferrous and ferric ions on the formation of trihalomethanes (THMs) and haloacetic acids (HAAs) were investigated using natural organic matter (NOM), small molecular weight NOM surrogates and natural water samples. The results showed that the effects were greatly dependent on the disinfection byproduct (DBP) precursor structure and molecular weight, and metal ions species. While using NOM as precursors, addition of 4.00 mM calcium ions increased the formation of THMs, dihaloacetic acids (DHAAs) and trihaloacetic acids (THAAs) by 24-47%, 51-61% and 15-25%, respectively. Addition of cupric ions at 0.02 mM increased the formation of THMs and DHAAs by 74-83% and 90-100%, respectively, but decreased the formation of THAAs by 26-27%. Similar effect was not observed when 0.04 mM ferrous or ferric ions were added. The effects of calcium and cupric ions on DBP formation were generally more evident for the NOM surrogates than that for NOM. The primary catalytic effect of calcium ions was due to complexation and less sensitive to molecular structure or weight, while that of cupric ions was attributed to redox reactions and greatly dependent on molecular structure. Both ferric and ferrous iron had substantial effects on the DBP formation of surrogates (citric acid and catechol in particular), which implied that the catalytic effects of ferric and ferrous iron mainly depended on molecular weight. The catalytic effect of cupric ions was also observed on natural water samples, while the effects of calcium, ferrous and ferric ions on natural water samples were not evident.

Afatinib reverses multidrug resistance in ovarian cancer via dually inhibiting ATP binding cassette subfamily B member 1.

ABCB1-mediated multidrug resistance (MDR) remains a major obstacle to successful chemotherapy in ovarian cancer. Herein, afatinib at nontoxic concentrations significantly reversed ABCB1-mediated MDR in ovarian cancer cells in vitro (p < 0.05). Combining paclitaxel and afatinib caused tumor regressions and tumor necrosis in A2780T xenografts in vivo. More interestingly, unlike reversible TKIs, afatinib had a distinctive dual-mode action. Afatinib not only inhibited the efflux function of ABCB1, but also attenuated its expression transcriptionally via down-regulation of PI3K/AKT and MAPK/p38-dependent activation of NF-κB. Furthermore, apart from a substrate binding domain, afatinib could also bind to an ATP binding domain of ABCB1 through forming hydrogen bonds with Gly533, Gly534, Lys536 and Ala560 sites. Importantly, mutations in these four binding sites of ABCB1 and the tyrosine kinase domain of EGFR were not correlated with the reversal activity of afatinib on MDR. Given that afatinib is a clinically approved drug, our results suggest combining afatinib with chemotherapeutic drugs in ovarian cancer. This study can facilitate the rediscovery of superior MDR reversal agents from molecular targeted drugs to provide a more effective and safer way of resensitizing MDR.

Protective effect of Huang Gan formula in 5/6 nephrectomized rats by depressing the Wnt/ß-catenin signaling pathway.

Huang Gan formula (HGF) is a new traditional Chinese herbal medicine created according to the basic theory of traditional Chinese medicine. The aim of this study is to evaluate the effects of HGF on chronic kidney disease and determine the mechanisms of action. The extract of HGF was prepared, and qualitative and quantitative determination of phytochemical was performed with quadrupole time-of-flight mass spectrometer and high-performance liquid chromatography. Sprague-Dawley rats (n=72) were submitted to 5/6 nephrectomy (Nx), and then respectively treated with uremic clearance granule, losartan, HGF low dose, HGF middle dose, and HGF high dose once per day for 12 weeks. The sham group of operated rats (n=22) was treated with normal saline or HGF middle dose as a background control group. Blood and urine biochemical parameters, renal tissue morphology, and mRNA and proteins of Wnt/ß-catenin signaling pathways were investigated. The results showed that the quality of the extraction process could be controlled, and a total of eight major compounds were identified and quantified. HGF could decrease the level of serum creatinine, blood urea nitrogen, and urine protein and increase the renal index and creatinine clearance rate in a dose-dependent manner. HGF also remarkably reduced the glomerulosclerosis and tubulointerstitial fibrosis by blocking the Wnt/ß-catenin signaling pathway through inhibiting the Wnt1, ß-catenin, transcription factor 4, and fibronectin 1 expressions, simultaneously measured through mRNA and protein levels in the remnant kidney. These results suggest that extraction of HGF could improve remnant renal function and possibly ameliorate glomerulosclerosis and tubulointerstitial fibrosis by depressing the Wnt/ß-catenin signaling pathway.

Microarray: a global analysis of biomineralization-related gene expression profiles during larval development in the pearl oyster, Pinctada fucata.

BACKGROUND: The molluscan Pinctada fucata is an important pearl-culturing organism to study biomineralization mechanisms. Several biomineralization-related genes play important roles regulating shell formation, but most previous work has focused only on their functions in adult oysters. Few studies have investigated biomineralization during larval development, when the shell is initially constructed and formed until the juvenile stage in dissoconch shells. Here, we report, for the first time, a global gene analysis during larval development of P. fucata based on a microarray and reveal the relationships between biomineralization-related genes and the shell formation process. RESULTS: Based on the P. fucata mantle transcriptome, 58,940 probes (60 nt), representing 58,623 transcripts, were synthesized. The gene expression profiles of the fertilized egg, trochophore, D-shaped, and umbonal stage larvae, as well as juveniles were analyzed by microarray performance. The expression patterns of the biomineralization-related genes changed corresponding to their regulatory function during shell formation. Matrix proteins chitin synthase and PFMG2 were highly expressed at the D-shaped stage, whereas PFMG6, PFMG8 and PfN23 were significantly up-regulated at the umbonal stage, indicating different roles regulating the formation of either periostracum, Prodissoconch I or Prodissoconch II shells. However, the majority of matrix proteins were expressed at high levels at the juvenile stage, and the shells comprised both an aragonitic nacreous layer and a calcitic prismatic layer as adults. We also identified five new genes that were significantly up-regulated in juveniles. These genes were expressed particularly in the mantle and coded for secreted proteins with tandem-arranged repeat units, as most matrix proteins. RNAi knockdown resulted in disrupted nacreous and prismatic shell layers, indicating their potential roles in shell formation. CONCLUSIONS: Our results add a global perspective on larval expression patterns of P. fucata genes and propose a mechanism of how biomineralization-related genes regulate the larval shell formation process. These results increase knowledge about biomineralization-related genes and highlight new aspects of shell formation mechanisms.

Differential gene expression profiling of Actinobacillus pleuropneumoniae during induction of primary alveolar macrophage apoptosis in piglets.

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is the causative agent of porcine pleuropneumonia, a disease that causes serious problems for the swine industry. Successful infection by this bacterium requires breaking the first line of defence in the lungs, the primary alveolar macrophages (PAMs). Therefore, exploring A. pleuropneumoniae-PAM interactions will provide vital groundwork for the scientific control of this infectious disease, which has been little studied up to now. In this work, PAMs were isolated from piglets and co-incubated with A. pleuropneumoniae serovar 5b strain L20 in vitro, and their interaction, PAM cell death, and differential gene expression of A. pleuropneumoniae in response to PAM cell death were observed and analysed using confocal microscopy, electron microscopy, RT-PCR, Western blot, flow cytometry and the use of a gene expression profile chip. A. pleuropneumoniae quickly adhered to and invaded PAMs, inducing apoptosis, which was confirmed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The highest percentage of apoptosis in cells was confirmed using flow cytometry when the cells were infected at a multiplicity of infection (MOI) of 10 and incubated for 5 h, with higher expression of activated caspase-3 as measured by Western blot. Using microarray gene chips with 2868 probes containing nearly all of the genomic sequence of A. pleuropneumoniae serotype 5b strain L20, a total of 185 bacterial genes were found to be differentially expressed (including 92 up-regulated and 93 down-regulated genes) and involved in the process of apoptosis, as compared with the expression of control bacteria cultured without PAMs in BHI medium (mean expression ratios >1.5-fold, p < 0.05). The up-regulated genes are involved in energy metabolism, gene transcription and translation, virulence related gene such as LPS, Trimeric Autotransporter Adhesin, RTX and similar genes. The down-regulated genes are involved in amino acid, cofactor, and vitamin metabolism, and also include ABC transporters. These data demonstrate that A. pleuropneumoniae induces apoptosis of PAMs and undergoes complex changes in gene transcription, including expression changes in known and potential virulence factors. Some potentially novel virulence targets have been identified, suggesting new strategies for the development of vaccines and medicines for both preventive and clinical use.

A novel conditioning regimen improves outcomes in ß-thalassemia major patients using unrelated donor peripheral blood stem cell transplantation.

We used a novel NF-08-TM transplant protocol based on intravenous busulfan, cyclophosphamide, fludarabine, and thiotepa in 82 consecutive patients with ß-thalassemia major (TM), including 52 with allogeneic peripheral blood stem cell transplantation (PBSCT) from unrelated donors (UDs) with well-matched human leukocyte antigens and 30 with hematopoietic stem cell transplantation (HSCT) from matched sibling donors (MSDs). The median age at transplantation was 6.0 years (range, 0.6-15.0 years), and the ratio of male-to-female patients was 56:26. The median follow-up time was 24 months (range, 12-39 months). The estimated 3-year overall survival and TM-free survival were 92.3% and 90.4% in the UD-PBSCT group and 90.0% and 83.3% in the MSD-HSCT group. The cumulative incidences of graft rejection and grades III-IV acute graft-versus-host disease were 1.9% and 9.6%, respectively, in the UD-PBSCT group and 6.9% and 3.6%, respectively, in the MSD-HSCT group. The cumulative incidence of transplant-related mortality was 7.7% in the UD-PBSCT group and 10.0% in the MSD-HSCT group. In conclusion, UD-PBSCTs using the well-tolerated NF-08-TM protocol show similar results to MSD-HSCTs and can be used to treat ß-thalassemia patients in the absence of MSDs.

[Pharmacokinetics of amphotericin B in the cerebrospinal fluid during continuous intrathecal administration for treatment of cryptococcal neoformans meningitis].

OBJECTIVE: To explore the pharmacokinetics of amphotericin B (AMB) in the cerebrospinal fluid (CSF) during continuous intrathecal administration of AMB for treatment of cryptococcal neoformans meningitis (CNM). METHODS: The concentration of AMB in the CSF was measured using reversed phase high performance liquid chromatography (RP-HPLC) in 3 patients receiving continuous intrathecal infusion of AMB for CNM. RESULTS: AMB concentrations in the CSF of the 3 patients exceeded the minimal inhibitory concentration (MIC) of AMB against Cryptococcus neoformans. The concentration-time curve showed that AMB concentration in the CSF underwent obvious variations on the first day of intrathecal infusion and after additional AMB doses, but maintained a stable level (0.61-1.21 µg/ml) on the next day. CONCLUSION: [corrected] Continuous intrathecal administration of AMB can enhance the drug concentration in the CSF and maintain a stable and effective drug level for treatment of CNM.