Assessing biochar, clinoptilolite zeolite and zeo-char loaded nano-nitrogen for boosting growth performance and biochemical ingredients of peace lily (Spathiphyllum Wallisii) plant under water shortage.

BACKGROUND: Peace lily (Spathiphyllum wallisii Regel) is an ornamental indoor plant with promising cut flower market, as well as antiviral, pharmacological and ecological potentials. Water deficiency can have sound effects on the growth performance and aesthetic quality of such plant. The aim of this study was to investigate the consequences of zeolite, biochar, and zeo-char loaded nano-nitrogen application on the growth performance and biochemical components of peace lily under water shortage conditions. An experiment was conducted over two consecutive seasons (2021-2022) at the experimental nursery of Ornamental Horticulture Department, Faculty of Agriculture, Cairo University, Giza, Egypt. Soil amendments; zeolite, biochar, and zeo-char loaded nano-nitrogen were prepared and applied to soil before cultivation. RESULTS: Our results revealed that the new combination treatment (zeo-char loaded nano-N) had an exceeding significant effect on most of the studied parameters. Vegetative traits such as plant height (35.7 and 35.9%), leaf number per plant (73.3 and 52.6%), leaf area (40.2 and 36.4%), stem diameter (28.7 and 27.1%), root number (100 and 43.5%) and length (105.7 and 101.9%) per plant, and fresh weight of leaves (23.2 and 21.6%) were significantly higher than control (commercially recommended dose of NPK) with the application of zeo-char loaded nano-N during the two growing seasons, respectively. Similar significant increments were obtained for some macro- (N, P, K, Mg, Ca) and micro- (Fe, Zn, Mn) elements with the same treatment relative to control. Chlorophyll (18.4%) and total carotenoids (82.9 and 32.6%), total carbohydrates (53.3 and 37.4%), phenolics (54.4 and 86.9%), flavonoids (31.7% and 41.8%) and tannins (69.2 and 50%), in addition to the phytohormone gibberellic acid (GA3) followed the same trend with the application of zeo-char loaded nano-N, increasing significantly over control. Leaf histological parameters and anatomical structure were enhanced with the new combination treatment in comparison with control. Antioxidant enzymes (catalase and peroxidase), proline and abscisic acid (ABA) exhibited significant declines with zeo-char loaded nano-N treatment relative to control. CONCLUSION: These findings suggest that incorporating soil amendments with nano- nutrients could provide a promising approach towards improving growth performance and quality of ornamental, medicinal and aromatic species under water deficiency conditions.

In vivo testing of novel nitric oxide-releasing nanoparticles for alleviating heart failure using the zebrafish embryo model.

Heart failure (HF) is a multifactorial, heterogeneous systemic disease that is considered one of the leading causes of death and morbidity worldwide. It is well-known that endothelial dysfunction (ED) plays an important role in cardiac disease etiology. A reduction in the bioavailability of nitric oxide (NO) in the bloodstream leads to vasoconstriction and ED. Many studies indicated diminishment of peripheral arteries vasodilation that is mediated by the endothelium in the of patients with chronic HF. With the advancement of nanomedicine, nanotechnology can provide adequate solutions for delivering exogenous NO with the aid of nanoparticles (NPs) to treat ED. The properties of superparamagnetic iron oxide nanoparticles (SPIONs) enable both passive and active delivery of drugs. This prompted us to investigate the efficacy of our newly-developed hydrogel nanoparticles (NO-RPs) for the delivery and sustained release of NO gas to alleviate cardiac failure and inflammation in the heart failure zebrafish model. The hydrogel NO-RPs incorporate SPIONS and NO precursor. The sustainend release of NO in the NO-RPs (4200 s), overcomes the problem of the short half life of NO in vivo which is expected to ameliorate the reduced NO bioavailabilty, and its consequences in endothelial and cardiac dysfunction. Zebrafish embryos were used as the animal model in this study to determine the effect of SPIONs-loaded NO-RPs on the cardiovascular system. Cardiac failure was induced in 24hpf embryos by exposure to aristolochic acid (AA)(0.25, 0.5 µM) for 8 h, followed by the SPIONs-loaded NO-RPs (0.25, 0.5 mg/ml) for 48 h, experimental groups included: control group which is healthy non treated zebrafish embryos, AA injured zebrafish embryos (HF) model,and NO-RP treated HF zebrafish embryos. Survival rate was assessed at 72hpf. Cardiac function was also evaluated by analyzing cardiac parameters including heartbeat, major blood vessels primordial cardinal vein and dorsal aorta (PCV &DA) diameter, blood flow velocity in PCV & DA vessels, cardiac output, and PCV & DA shear stresses. All cardiac parameters were analyzed with the aid of MicroZebraLab blood flow analysis software from Viewpoint. In addition, we studied the molecular effects of the developed NO-RPs on the mRNA expression of selected pro-inflammatory markers: IL-6, and Cox-2. Our findings demonstrated that the NO-RPs improved the survival rate in the heart failure zebrafish model and reversed heart failure by enhancing blood flow perfusion in Zebrafish embryos, significantly. In addition, RT-PCR results showed that the NO-RPs significantly reduced the expression of pro-inflammatory markers (lL-6&COX-2) in the heart failure zebrafish model. Our study confirmed that the developed SPIONs-loaded NO-RPs are effective tool to alleviate cardiac failure and inflammation in the HF zebrafish model.

Gender-based effect of absence of gut microbiota on the protective efficacy of Bifidobacterium longum-fermented rice bran diet against inflammation-associated colon tumorigenesis.

Dietary rice bran (RB) has shown capacity to influence metabolism by modulation of gut microbiota in individuals at risk for colorectal cancer (CRC), which warranted attention for delineating mechanisms for bidirectional influences and cross-feeding between the host and RB-modified gut microbiota to reduce CRC. Accordingly, in the present study, fermented rice bran (FRB, fermented with a RB responsive microbe Bifidobacterium longum), and non-fermented RB were fed as 10% w/w (diet) to gut microbiota-intactspf or germ-free micegf to investigate comparative efficacy against inflammation-associated azoxymethane/dextran sodium sulfate (AOM/DSS)-induced CRC. Results indicated both microbiota-dependent and independent mechanisms for RB meditated protective efficacy against CRC that was associated with reduced neoplastic lesion size and local-mucosal/systemic inflammation, and restoration of colonic epithelial integrity. Enrichment of beneficial commensals (such as, Clostridiales, Blautia, Roseburia), phenolic metabolites (benzoate and catechol metabolism), and dietary components (ferulic acid-4 sulfate, trigonelline, and salicylate) were correlated with anti-CRC efficacy. Germ-free studies revealed gender-specific physiological variables could differentially impact CRC growth and progression. In the germ-free females, the RB dietary treatment showed a ∼72% reduction in the incidence of colonic epithelial erosion when compared to the ∼40% reduction in FRB-fed micegf . Ex vivo fermentation of RB did not parallel the localized-protective benefits of gut microbial metabolism by RB in damaged colonic tissues. Findings from this study suggest potential needs for safety considerations of fermented fiber rich foods as dietary strategies against severe inflammation-associated colon tumorigenesis (particularly with severe damage to the colonic epithelium).

A Randomized Controlled Trial of Dietary Rice Bran Intake on Microbiota Diversity, Enteric Dysfunction, and Fecal Secretory IgA in Malian and Nicaraguan Infants.

BACKGROUND: Malnutrition and diarrhea are leading causes of death in children aged <5 y. Rice bran is a nutrient-dense prebiotic available globally. OBJECTIVES: The objective of this secondary analysis was to evaluate the effects of daily rice bran supplementation on environmental enteric dysfunction (EED) markers, total fecal secretory IgA (sIgA), and microbiota in infants at high risk of malnutrition. METHODS: Six-month-old Malian and Nicaraguan infants were randomly assigned to control or daily rice bran supplementation cohorts (1 to 5 g/d). Feces were collected monthly for 6 mo to evaluate fecal sIgA, markers of EED, and microbiota diversity. Statistical methods included linear mixed models, generalized mixed models, Spearman correlation, and Wilcoxon rank-sum tests. RESULTS: Six-month-old Malian infants had significantly elevated sIgA (4.0× higher, P < 0.001), fecal myeloperoxidase (31.6× higher, P < 0.001), fecal α1-antitrypsin (1.8× higher, P = 0.006), and lower fecal neopterin (0.13× higher, P < 0.001) than the age-matched Nicaraguan infants. In the Nicaraguan rice bran cohort from 6 to 12 mo of age, there was a significant decrease in sIgA concentrations (0.4×, P < 0.05) and a correlation between sIgA and the EED marker α1-antitrypsin (0.523, P < 0.0001) at 12 mo of age. In Malian infants, daily rice bran ingestion resulted in decreased EED scores (0.71×, P = 0.02) and a stable sIgA concentration over time. The rice bran group of Malian infants also had correlation between sIgA and the EED marker neopterin (0.544, P < 0.001) at 12 mo of age and a significant (P < 0.05) increase in microbiota α-diversity at a younger age (9 mo with rice bran compared with 10 mo in control group), which supports earlier microbiota maturation. CONCLUSIONS: These results support rice bran as a functional food ingredient targeting gut mucosa in children at high-risk of malnutrition.

In Silico/In Vitro Hit-to-Lead Methodology Yields SMYD3 Inhibitor That Eliminates Unrestrained Proliferation of Breast Carcinoma Cells.

SMYD3 is a lysine methyltransferase that regulates the expression of over 80 genes and is required for the uncontrolled proliferation of most breast, colorectal, and hepatocellular carcinomas. The elimination of SMYD3 restores normal expression patterns of these genes and halts aberrant cell proliferation, making it a promising target for small molecule inhibition. In this study, we sought to establish a proof of concept for our in silico/in vitro hit-to-lead enzyme inhibitor development platform and to identify a lead small molecule candidate for SMYD3 inhibition. We used Schrodinger® software to screen libraries of small molecules in silico and the five compounds with the greatest predicted binding affinity within the SMYD3 binding pocket were purchased and assessed in vitro in direct binding assays and in breast cancer cell lines. We have confirmed the ability of one of these inhibitors, Inhibitor-4, to restore normal rates of cell proliferation, arrest the cell cycle, and induce apoptosis in breast cancer cells without affecting wildtype cell behavior. Our results provide a proof of concept for this fast and affordable small molecule hit-to-lead methodology as well as a promising candidate small molecule SMYD3 inhibitor for the treatment of human cancer.

Nucleophosmin deposition during mRNA 3' end processing influences poly(A) tail length.

During polyadenylation, the multi-functional protein nucleophosmin (NPM1) is deposited onto all cellular mRNAs analysed to date. Premature termination of poly(A) tail synthesis in the presence of cordycepin abrogates deposition of the protein onto the mRNA, indicating natural termination of poly(A) addition is required for NPM1 binding. NPM1 appears to be a bona fide member of the complex involved in 3' end processing as it is associated with the AAUAAA-binding CPSF factor and can be co-immunoprecipitated with other polyadenylation factors. Furthermore, reduction in the levels of NPM1 results in hyperadenylation of mRNAs, consistent with alterations in poly(A) tail chain termination. Finally, knockdown of NPM1 results in retention of poly(A)(+) RNAs in the cell nucleus, indicating that NPM1 influences mRNA export. Collectively, these data suggest that NPM1 has an important role in poly(A) tail length determination and may help network 3' end processing with other aspects of nuclear mRNA maturation.

Coimmobilization of l-methioninase and glutamate dehydrogenase: Novel approach for L-homoalanine synthesis.

L-Homoalanine, a nonnatural amino acid that is rarely found in human and microorganisms, is used in the synthesis of various medically pivotal antiepileptic drugs and antituberculosis compounds. l-Homoalanine can be synthesized by different enzymatic approaches. In this article, the synthesis of l-homoalanine from l-methionine was explored by coimmobilization of Aspergillus flavipes l-methioninase (AfMETase) and glutamate dehydrogenase (GDH) on polyacrylamide and chitosan. Polyacrylamide coimmobilized AfMETase and GDH displayed a maximum reactivity for the synthesis of homoalanine from l-methionine. The chitosan-coimmobilized AfMETase and GDH retain about 70% of their initial activity of l-homoalanine production by the fifth catalytic reusability cycle as compared with 50% for polyacrylamide coimmobilizate. Catalytic conditions were optimized for the maximum yield of homoalanine. Homoalanine was purified by cationic and anionic chromatographs and the proton nuclear magnetic resonance (H-NMR) analysis of the lyophilized sample displayed a unique chemical structure identical to the authentic homoalanine. Using dependable dual action of AfMETase and GDH immobilized on a solid support is a novel approach for in vitro enzymatic synthesis of l-homoalanine from l-methionine, and the immobilized enzymes can be reused many times without any significant loss of their activities.

Integrated Microbiota and Metabolite Changes following Rice Bran Intake during Murine Inflammatory Colitis-Associated Colon Cancer and in Colorectal Cancer Survivors.

Dietary rice bran-mediated inhibition of colon carcinogenesis was demonstrated previously for carcinogen-induced rodent models via multiple anti-cancer mechanisms. This study investigated the role of dietary rice bran-mediated changes to fecal microbiota and metabolites over the time course of colon carcinogenesis and compared murine fecal metabolites to human stool metabolic profiles following rice bran consumption by colorectal cancer survivors (NCT01929122). Forty adult male BALB/c mice were subjected to azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colitis-associated colon carcinogenesis and randomized to control AIN93M (n = 20) or diets containing 10% w/w heat-stabilized rice bran (n = 20). Feces were serially collected for 16S rRNA amplicon sequencing and non-targeted metabolomics. Fecal microbiota richness and diversity was increased in mice and humans with dietary rice bran treatment. Key drivers of differential bacterial abundances from rice bran intake in mice included Akkermansia, Lactococcus, Lachnospiraceae, and Eubacterium xylanophilum. Murine fecal metabolomics revealed 592 biochemical identities with notable changes to fatty acids, phenolics, and vitamins. Monoacylglycerols, dihydroferulate, 2-hydroxyhippurate (salicylurate), ferulic acid 4-sulfate, and vitamin B6 and E isomers significantly differed between rice bran- and control-fed mice. The kinetics of murine metabolic changes by the host and gut microbiome following rice bran consumption complemented changes observed in humans for apigenin, N-acetylhistamine, and ethylmalonate in feces. Increased enterolactone abundance is a novel diet-driven microbial metabolite fecal biomarker following rice bran consumption in mice and humans from this study. Dietary rice bran bioactivity via gut microbiome metabolism in mice and humans contributes to protection against colorectal cancer. The findings from this study provide compelling support for rice bran in clinical and public health guidelines for colorectal cancer prevention and control.

Plasma, urine, and stool metabolites in response to dietary rice bran and navy bean supplementation in adults at high-risk for colorectal cancer.

Introduction: Dietary intake of whole grains and legumes and adequate physical activity (PA) have been associated with reduced colorectal cancer (CRC) risk. A single-blinded, two-arm, randomized, placebo-controlled pilot trial was implemented to evaluate the impact of a 12-week dietary intervention of rice bran + navy bean supplementation and PA education on metabolite profiles and the gut microbiome among individuals at high risk of CRC. Methods: Adults (n=20) were randomized 1:1 to dietary intervention or control. All participants received PA education at baseline. Sixteen study foods were prepared with either heat-stabilized rice bran + navy bean powder or Fibersol®-2 as a placebo. Intervention participants consumed 30 g rice bran + 30 g navy bean powder daily; those in the control group consumed 10 g placebo daily. Non-targeted metabolite profiling was performed by UPLC-MS/MS to evaluate plasma, urine, and stool at 0, 6, and 12 weeks. Stool was also analyzed for primary and secondary bile acids (BAs) and short chain fatty acids (SCFAs) by UPLC-MS/MS and microbial community structure via 16S amplicon sequencing. Two-way ANOVA was used to compare differences between groups for metabolites, and mixed models were used to compare differences between groups for BAs, SCFAs, and alpha and beta diversity measures of microbial community structure. Results: Across biological matrices, the intervention resulted in changes to several amino acid and lipid metabolites, compared to control. There was a 2.33-fold difference in plasma (p<0.001) and a 3.33-fold difference in urine (p=0.008) for the amino acid S-methylcysteine at 12 weeks. Fold-differences to 4-methoxyphenol sulfate in plasma and urine after 6 and 12 weeks (p<0.001) was a novel result from this combined rice bran and navy bean intervention in people. A 2.98-fold difference in plasma (p=0.002) and a 17.74-fold difference in stool (p=0.026) was observed for the lipid octadecenedioylcarnitine at 12 weeks. For stool BAs, 3-oxocholic acid was increased at 12 weeks compared to control within a subset of individuals (mean difference 16.2 ug/uL, p=0.022). No significant differences were observed between groups for stool SCFAs or microbial community structure. Discussion: Dietary intake of rice bran + navy beans demonstrates beneficial modulation of host and gut microbial metabolism and represents a practical and affordable means of increasing adherence to national guidelines for CRC control and prevention in a high-risk population.

Non-Targeted Dried Blood Spot-Based Metabolomics Analysis Showed Rice Bran Supplementation Effects Multiple Metabolic Pathways during Infant Weaning and Growth in Mali.

Rice bran contains essential nutrients, antioxidants, and bioactives with anti-inflammatory and diarrheal protective properties important for infants. This 6-month randomized controlled trial investigated the effects of heat-stabilized rice bran supplementation during Malian infant weaning. Fifty healthy 6-month-old infants were randomized to a rice bran intervention (N = 25) or non-intervention control group (N = 25). Intervention infants received dose-escalating rice bran supplementation for 6 months (1-5 g/day). Monthly infant dried blood spot and anthropometric measurements were collected. Dried blood spot metabolite abundances were compared monthly according to diet for six months. Supplementation resulted in favorable weight-for-age and weight-for-length z-score changes. Non-targeted dried blood spot-based metabolomics identified 796 metabolites, of which 33% had significant fold differences between groups (7-12 months). Lipids and amino acids represented 70.6% of the metabolites identified. Rice bran supplementation during infant weaning significantly modulated the metabolites involved in antioxidant defenses and with neuroactive properties including reduced glutathione, glycine, glutamate, cysteinylglycine, tryptophan betaine, and choline. These findings support rice bran as a weaning ingredient to meet infant nutritional requirements and with the potential to reduce oxidative stress and improve cognitive outcomes. This study provides evidence for dried blood spots as a cost-effective tool to detect infant biomarkers of nutritional and metabolic status.

Dietary Rice Bran-Modified Human Gut Microbial Consortia Confers Protection against Colon Carcinogenesis Following Fecal Transfaunation.

Rice bran, removed from whole grain rice for white rice milling, has demonstrated efficacy for the control and suppression of colitis and colon cancer in multiple animal models. Dietary rice bran intake was shown to modify human stool metabolites as a result of modifications to metabolism by gut microbiota. In this study, human stool microbiota from colorectal cancer (CRC) survivors that consumed rice bran daily was examined by fecal microbiota transplantation (FMT) for protection from azoxymethane and dextran sodium sulfate (AOM/DSS) induced colon carcinogenesis in germ-free mice. Mice transfaunated with rice bran-modified microbiota communities (RMC) harbored fewer neoplastic lesions in the colon and displayed distinct enrichment of Flavonifractor and Oscillibacter associated with colon health, and the depletion of Parabacteroides distasonis correlated with increased tumor burden. Two anti-cancer metabolites, myristoylcarnitine and palmitoylcarnitine were increased in the colon of RMC transplanted mice. Trimethylamine-N-oxide (TMAO) and tartarate that are implicated in CRC development were reduced in murine colon tissue after FMT with rice bran-modified human microbiota. Findings from this study show that rice bran modified gut microbiota from humans confers protection from colon carcinogenesis in mice and suggests integrated dietary-FMT intervention strategies should be tested for colorectal cancer control, treatment, and prevention.

RNA recognition by 3'-to-5' exonucleases: the substrate perspective.

The 3'-to-5' exonucleolytic decay and processing of a variety of RNAs is an essential feature of RNA metabolism in all cells. The 3'-5' exonucleases, and in particular the exosome, are involved in a large number of pathways from 3' processing of rRNA, snRNA and snoRNA, to decay of mRNAs and mRNA surveillance. The potent enzymes performing these reactions are regulated to prevent processing of inappropriate substrates whilst mature RNA molecules exhibit several attributes that enable them to evade 3'-5' attack. How does an enzyme perform such selective activities on different substrates? The goal of this review is to provide an overview and perspective of available data on the underlying principles for the recognition of RNA substrates by 3'-to-5' exonucleases.

Antimicrobial-Resistant Escherichia coli from Environmental Waters in Northern Colorado.

Waterborne Escherichia coli are a major reservoir of antimicrobial resistance (AMR), including but not limited to extended-spectrum beta-lactamase (ESBL) and Klebsiella pneumoniae carbapenemase (KPC) mechanisms. This study quantified and described ESBL- and KPC-producing E. coli in Northern Colorado from sewer water, surface water, and influent and effluent wastewater treatment sources. Total detected bacteria and E. coli abundances, and the percentages that contain ESBL and/or KPC, were compared between water sources. Seventy E. coli isolates from the various waters had drug resistance validated with a panel of 17 antibiotics using a broth microdilution assay. The diverse drug resistance observed across E. coli isolates was further documented by polymerase chain reaction of common ESBL genes and functional relatedness by PhenePlate assay-generated dendrograms (n=70). The total E. coli abundance decreased through the water treatment process as expected, yet the percentages of E. coli harboring ESBL resistance were increased (1.70%) in surface water. Whole-genome sequencing analysis was completed for 185 AMR genes in wastewater E. coli isolates and confirmed the presence of diverse AMR gene classes (e.g., beta-lactams and efflux pumps) in isolate genomes. This study completed surveillance of AMR patterns in E. coli that reside in environmental water systems and suggests a role for integrating both phenotypic and genotypic profiling beyond ESBL and KPC mechanisms. AMR screening via multiple approaches may assist in the prevention of drug-resistant E. coli spread from waters to animals and humans.

Rice bran supplementation modulates growth, microbiota and metabolome in weaning infants: a clinical trial in Nicaragua and Mali.

Rice bran supplementation provides nutrients, prebiotics and phytochemicals that enhance gut immunity, reduce enteric pathogens and diarrhea, and warrants attention for improvement of environmental enteric dysfunction (EED) in children. EED is a subclinical condition associated with stunting due to impaired nutrient absorption. This study investigated the effects of rice bran supplementation on weight for age and length for age z-scores (WAZ, LAZ), EED stool biomarkers, as well as microbiota and metabolome signatures in weaning infants from 6 to 12 months old that reside in Nicaragua and Mali. Healthy infants were randomized to a control (no intervention) or a rice bran group that received daily supplementation with increasing doses at each month (1-5 g/day). Stool microbiota were characterized using 16S rDNA amplicon sequencing. Stool metabolomes were analyzed using ultra-high-performance liquid-chromatography tandem mass-spectrometry. Statistical comparisons were completed at 6, 8, and 12 months of age. Daily consumption of rice bran was safe and feasible to support changes in LAZ from 6-8 and 8-12 months of age in Nicaragua and Mali infants when compared to control. WAZ was significantly improved only for Mali infants at 8 and 12 months. Mali and Nicaraguan infants showed major differences in the overall gut microbiota and metabolome composition and structure at baseline, and thus each country cohort demonstrated distinct microbial and metabolite profile responses to rice bran supplementation when compared to control. Rice bran is a practical dietary intervention strategy that merits development in rice-growing regions that have a high prevalence of growth stunting due to malnutrition and diarrheal diseases. Rice is grown as a staple food, and the bran is used as animal feed or wasted in many low- and middle-income countries where EED and stunting is prevalent.

Newly-established free water-surface constructed wetland to treat agricultural waters in the low-lying Venetian plain: Performance on nitrogen and phosphorus removal.

Constructed wetlands offer promising solutions for controlling nutrient pollution in agricultural systems with relatively low costs and energy inputs. In mainly central and northern Italy, semi-natural and reconstructed Free-Water Surface Constructed Wetlands (FWS CWs) are designed to treat nonpoint-source pollution from agricultural catchments. However, their performance depends on system design and time of establishment. This paper evaluates the efficiency of a recently established FWS CW to remove nonpoint-source nutrient pollution due to agricultural drainage in the low-lying Venetian plain (NE Italy). The system was established in 2014 by creating five consecutive sub-basins vegetated with macrophytes to restore a semi-natural wetland, and later monitored in terms of water quality parameters and nutrients removal over three consecutive agricultural seasons (2014-2016). Total (TN) and nitrate (N-NO3) nitrogen concentrations showed peaks (16.37 and 15.31 mg l-1 for TN and N-NO3, respectively) in the various sub-basins during spring 2015, associated with fertilisation of surrounding croplands and intense rain events. Performance improved over the three years, with increasing median removals of TN (33.3-49.0%) and N-NO3 (32.2-80.5%), corresponding to average mass of 1355 kg y-1 and 1011 kg y-1 for TN and N-NO3. Concentrations of ammonium (N-NH4) and orthophosphate (P-PO4) were generally low (<1 and <0.3 mg l-1 for N-NH4 and P-PO4, respectively), with average yearly mass removals of 50 kg for N-NH4 and 9 kg for P-PO4. According to the overall treatment performance, the FWS CW could treat a total area of about 30 ha with a wetland/catchment ratio of 7%. However, we expect that treatment efficiency will increase as a result of bank stabilisation and improvement of the aquatic environment, together with increases in surface vegetation.

Clinical study of different composite resin systems in Class I cavities (an 18-month randomized clinical trial) / Estudo clínico de diferentes sistemas de resina composta em cavidades de Classe I (um ensaio clínico randomizado de 18 meses)

Objective: this double-blind randomized clinical trial evaluate the clinical performance of Thermo-Viscous Bulk Fill composite, Self-Adhesive Flowable composite, and Filtek Bulk Fill Composites restorations in Class I cavities over a period of 18 months. Material and Methods: twenty individuals between the ages of 30 and 45 participated in this research. Each patient should have at least three occlusal Class I carious lesions on their molars. They were dispersed at random, with n=20 teeth representing each tested material. Group I (Futurabond M+ and VisCalor Bulk Fill which heated in a viscolar dispenser at 68 °C for 30s and placed in a 4 mm thickness), Group II (Fusio Liquid Dentin self-adhesive composite which put in a thin layer (1mm increment)), and Group III (Single Bond Universal and Filtek Bulk Fill Posterior composite which applied in 4 mm thickness without heating). Using (USPHS) criteria, all restorations were assessed clinically at baseline, 6 months, 12 months, and 18 months. Using an inverse replica, the marginal seal of the investigated restorations was further evaluated under SEM. Statistical analysis was performed with Chi-square test for all USPH parameters. Results: the three tested groups recorded a one hundred percent retention rate after 18 months follow up period. Concerning marginal adaptation, marginal discoloration, anatomical form, surface texture, and color matching, there was a significant difference (p˂0.05) between the three tested groups after 12 & 18 months. After 12 & 18 months, SEM analysis of the marginal seal revealed a statistically significant difference between the three groups. Conclusion: Bulk fill resin composite restorations showed satisfactory acceptable clinical performance after 18 months of clinical service compared to self-adhesive flowable composites, and Viscalor Bulk Fill composite demonstrated excellent results with considerable changes in marginal integrity as a consequence of thermal viscous technology and increased adaptability of restorations toward cavity walls and margins (AU)

Objetivo: este ensaio clínico randomizado duplo-cego avaliou o desempenho clínico de restaurações de resina Bulk Fill Termo-Viscosa, resina autoadesiva Flowable e Filtek Bulk Fill Composites em cavidades Classe I durante um período de 18 meses. Material e Métodos: 20 indivíduos com idade entre 30 e 45 anos participaram da pesquisa. Cada paciente deveria ter pelo menos três lesões de cárie oclusais de Classe I nos molares. Eles foram divididos aleatoriamente, com n=20 dentes representando cada material testado. Grupo I (Futurabond M+ e VisCalor Bulk Fill aquecido em dispensador viscolar a 68 °C por 30s e colocado em uma espessura de 4 mm), Grupo II (resina composta autoadesiva Fusio Liquid Dentin colocada em uma camada fina (incremento de 1 mm)) e Grupo III (resina composta Single Bond Universal e Filtek Bulk Fill Posterior aplicado em espessura de 4 mm sem aquecimento). Usando os critérios (USPHS), todas as restaurações foram avaliadas clinicamente no início, 6 meses, 12 meses e 18 meses. Usando uma réplica inversa, o selamento marginal das restaurações investigadas foi avaliado em MEV. A análise estatística foi realizada com o teste qui-quadrado para todos os parâmetros USPH. Resultados: os três grupos testados registraram uma taxa de retenção de cem por cento após um período de acompanhamento de 18 meses. Em relação à adaptação marginal, descoloração marginal, forma anatômica, textura da superfície e combinação de cores, houve uma diferença significativa (p˂0,05) entre os três grupos testados após 12 e 18 meses. Após 12 e 18 meses, a análise SEM do selamento marginal revelou uma diferença estatisticamente significativa entre os três grupos. Conclusão: as restaurações de resina composta Bulk Fill apresentaram desempenho clínico aceitável satisfatório após 18 meses de atendimento clínico em comparação com as resinas compostas fluidas autoadesivas, e a resina composta Viscalor Bulk Fill demonstrou excelentes resultados com mudanças consideráveis na integridade marginal, como consequência da tecnologia viscosa térmica e maior adaptabilidade de restaurações nas paredes e margens da cavidade (AU)

Biochemical stability and molecular dynamic characterization of Aspergillus fumigatus cystathionine γ-lyase in response to various reaction effectors.

Cystathionine γ-lyase (CGL) is a key enzyme in the methionine-cysteine cycle in all living organisms forming cysteine, α-ketobutyrate and ammonia via homocysteine and cystathionine intermediates. Although, human and plant CGLs have been extensively studied at the molecular and mechanistic levels, there has been little work on the molecular and catalytic properties of fungal CGL. Herein, we studied in detail for the first time the molecular and catalytic stability of Aspergillus fumigatus CGL, since conformational instability, inactivation and structural antigenicity are the main limitations of the PLP-dependent enzymes on various therapeutic uses. We examined these properties in response to buffer compositions, stabilizing and destabilizing agents using Differential Scanning Fluorometery (DSF), steady state and gel-based fluorescence of the intrinsic hydrophobic core, stability of internal aldimine linkage and catalytic properties. The activity of the recombinant A. fumigatus CGL was 13.8U/mg. The melting temperature (Tm) of CGL in potassium phosphate buffer (pH 7.0-8.0) was 73.3°C, with ∼3°C upshifting in MES and sodium phosphate buffers (pH 7.0). The conformational thermal stability was increased in potassium phosphate, sodium phosphate and MES buffers, in contrast to Tris-HCl, HEPES (pH 7.0) and CAPS (pH 9.0-10.0). The thermal stability and activity of CGL was slightly increased in the presence of trehalose and glycerol that might be due to hydration of the enzyme backbone, unlike the denaturing effect of GdmCl and urea. Modification of surface CGL glutamic and aspartic acids had no significant effect on the enzyme conformational and catalytic stability. Molecular modeling and dynamics simulations unveil the high conformational stability of the overall scaffold of CGL with high flexibility at the non-structural regions. CGL structure has eight buried Trp residues, which are reoriented to the enzyme surface and get exposed to the solvent under perturbation of destabilizers. Furthermore, electrostatic calculations of selected snapshots of CGL 3D structure under different experimental conditions showed a remarkable differences on the polarity of the enzyme surface.

Complexity and species variation of the kidney-type glutaminase gene.

Increased expression of rat kidney-type glutaminase (KGA) during metabolic acidosis results from selective mRNA stabilization. This process is mediated by an 8-base AU-sequence that functions as a pH-response element (pHRE). LLC-PK1-FBPase+ cells, a pH-responsive porcine kidney cell line, express four distinct GA mRNAs. RNase H mapping indicated that three of the GA mRNAs are generated by use of alternative polyadenylation sites and are homologs of the rat KGA mRNA, while the fourth contains a different COOH-terminal coding and 3'-untranslated sequence. PCR cloning and sequencing established that the latter GA mRNA is the homolog of the human GAC mRNA. A rat GAC cDNA was also cloned from a rat kidney library. The 3'-untranslated regions of the GAC mRNAs, but not the porcine or human KGA mRNAs, contain identifiable pHREs. The human KGA gene spans 82 kb and is composed of 19 exons. The unique sequence from the hGAC cDNA is contained in a single exon. Thus in humans, alternative splicing of the initial transcript could produce two GA mRNAs, only one of which may be increased during acidosis.

Co-immobilization of PEGylated Aspergillus flavipes L-methioninase with glutamate dehydrogenase: a novel catalytically stable anticancer consortium.

Aspergillus flavipes L-methioninase (AfMETase) exhibits reliable pharmacokinetic properties and anticancer potency in vitro[10]. To maximize its therapeutic efficiency as protection against in vivo proteolysis, reduction of antigenicity and hyperammoniemia, the enzyme was PEGylated and coupled with glutamate dehydrogenase (GDH). The highest degree of PEGylation was measured at 40-50/1 molar ratio of PEG to AfMETase, with a lower mobility on SDS-PGE, compared to the native AfMETase. The activity of free AfMETase was reduced to 66.2% and further to 50% upon PEGylation and GDH conjugation, respectively. The highest degree of surface NH2 modification of AfMETase-GDH co-immobilizates (65%), was reported using 300 mM glutaraldehyde, with 31% methionine conversion. Using L-cysteine and L-methionine as active site protectors, the activity of PEG-AfMETase and PEG-AfMETase-GDH was increased by 14.4 and 32.9-fold, respectively. At 45°C, PEG-AfMETase, PEG-AfMETase-GDH and AfMETase-GDH conjugate have a T1/2 10.3, 8.5 and 7.6 h, inactivation rate (Kr) 0.021, 0.03 and 0.016 min, with 2.0, 1.65 and 1.47-fold stabilization, respectively. Kinetically, the three immobilizates have a relatively similar Km values for L-methionine (7.4-7.9 mM), with lower affinity to homocysteine and cysteine, with stability to PLP-enzyme inhibitors (propargylglycine and hydroxylamine), indicating the protective effect by PEG moieties on the enzyme structure. Also, the three immobilizates exhibited improved stability against proteolysis in vitro, comparing to free AfMETase.

Role of deadenylation and AUF1 binding in the pH-responsive stabilization of glutaminase mRNA.

During chronic metabolic acidosis, increased expression of renal glutaminase (GA) results from selective stabilization of the GA mRNA. This response is mediated by a direct repeat of an 8-base adenylate-uridylate (AU) sequence that binds zeta-crystallin and functions as a pH response element (pH-RE). A tetracycline-responsive promoter system was developed in LLC-PK(1)-F(+) cells to perform pulse-chase analysis of the turnover of a chimeric beta-globin (betaG) mRNA that contains 960 bp of the 3'-UTR of GA mRNA including the pH-RE. The betaG-GA mRNA exhibits a 14-fold increase in half-life when the LLC-PK(1)-F(+) cells are transferred to acidic medium. RNase H cleavage and Northern blot analysis of the 3'-ends established that rapid deadenylation occurred concomitantly with the rapid decay of the betaG-GA mRNA in cells grown in normal medium. Stabilization of the betaG-GA mRNA in acidic medium is associated with a pronounced decrease in the rate of deadenylation. Mutation of the pH-RE within the betaG-GA mRNA blocked the pH-responsive stabilization, but not the rapid decay, whereas insertion of only a 29-bp segment containing the pH-RE was sufficient to produce both a rapid decay and a pH-responsive stabilization. Various kidney cells express multiple isoforms of AUF1, an AU-binding protein that enhances mRNA turnover. RNA gel-shift assays demonstrated that the recombinant p40 isoform of AUF1 binds to the pH-RE with high affinity and specificity. Thus AUF1 may mediate the rapid turnover of the GA mRNA, whereas increased binding of zeta-crystallin during acidosis may inhibit degradation and result in selective stabilization.