Lallemantia iberica (Bieb.) Fisch. and C. A. Mey is high valued annual ornamental and medicinal plant from Lamiaceae family that prefers dry sunny hillsides, roadsides, slopes, and fallow fields over an altitude of 500–2150 m. It bears beautiful white flowers and bloom from April to June each year. This study reports L. iberica micropropagation using cotyledon node explants isolated from 15-day-old in vitro regenerated plantlets. The cotyledon node explants were cultured on MS medium containing 0.50, 1.00 plus 2.00 mg/L BAP, 0.00, 0.01, and 0.02 mg/L NAA. Maximum shoot regeneration was noted on MS medium containing 0.50 mg/L BAP. Well-developed micropropagated shoots were rooted on MS medium containing 1.00 mg/L IBA. The rooted plants were easily hardened in the growth chamber and acclimatised in greenhouse. Lallemantia iberica, family Lamiaceae, is a beautiful ornamental annual plant that grows over large area in Turkey. The family contains 236 genera and about 7173 species that grow on wide area extending from the Asia minor to the Caucasian countries, the Middle East, and the Central and the Southern Europe over an altitude of 500–2150 m and blooms during April to June each year  supplemented with 3% (w/v) sucrose and 0.6%  (w/v) plant agar (Duchefa) for shoot regeneration and multiplication.The seeds were surface sterilized using 100% bleach  followed by rinsing 3 × 5 min with sterile distilled water. Thereafter, the seeds were cultured on agar solidified MS medium  containiWell-developed shoots induced on cotyledon node explant were used in the rooting experiment. These shoots were rooted on MS medium containing 1.00 mg/L IBA.−2. The pH of all media was adjusted to 5.7 ± 0.1 with 1 N NaOH or 1 N HCl. They were poured into 200 mm 10 × 10 mm culture boxes. All cultures were grown at 24 ± 2°C with a 16 h light photoperiod. Light was supplied at intensity of 45 μmol m−2 s−1 by Phillips day light lamps.All regeneration and rooting media were autoclaved for 20 min at 1.4 kg cmAfter acclimatization of well-rooted and healthy plantlets, agar was removed from their roots under running tap water followed by transfer to pots containing peat moss, which were covered with transparent polyethylene bags to create a high relative humidity during initial stages of prehardening and kept at 24° ± 1°C + 80% relative humidity in growth chamber. Each of the developing explants was transferred to 5.00 litre pots containing 4.50 litres of peat moss for 15–18 d. Thereafter, the transparent polyethylene bags were gradually opened and removed in 10–13 d after achieving hardening. These plantlets were maintained in greenhouse for growth, development, flowering, and morphological observations during posthardening or acclimatisation.Each replication contained 10 explants and each treatment was replicated thrice.X) transformation before statistical analysis following Snedecor and Cochran [b test using IBM SPSS for Windows v. 20.Experimental data given in percentages were subjected to square root . No callus induction was noted on MS medium containing 0.50 mg/L BAP . 100.00% shoot regeneration was noted on MS medium containing 0.5 mg/L BAP. The rest of the plant growth regulator combinations affected shoot regeneration in range of 33.33–86.67%. No shoot regeneration was noted on MS medium (control).The result showed that the combinations and rates of plant growth regulators used in the study sharply affected shoot induction percentage . Number of shoots per explant ranged from 3.33 to 17.80. Maximum shoot regeneration was noted on 0.5 mg/L BAP. However, these shoots were hyperhydric at the initial stages of growth  callusing was not noted on any regeneration medium. It varied variably due to interaction among physical-chemical factors affecting tissue cultured explants. The results are in agreement with He et al. , who notg et al.  used cal Portulaca grandiflora.This variation was more evident, when the results were related with the number of shoots per explant. Maximum number of shoots per explant was noted on MS medium containing 0.50 mg/L BAP with or without any concentration of NAA, when no callus was induced. Each increase in the BAP concentration was inhibitory and showed corresponding sharp decrease in the number of shoots per explant. MS medium containing 0.50, 1.00, and 2.00 mg/L BAP with 0.01 mg/L NAA had statistically similar effect on number of shoots per explant. Excluding this, the rest of the cultures induced 3.33 to 4.87 shoots per explant. The results of this study are in agreement with Patnaik and Debata , SmolensL. iberica cotyledon node explant reacted variably with BAP-NAA. Callusing was inhibited variably in the presence of NAA. However, concentrations of 1.00 and 2.00 mg/L BAP were equally good for callus induction and shoot regeneration. in vitro regenerated plantlets could be rooted and acclimatized without any difficulty. The results of this study are in agreement with Khawar et al. [ in vitro rooting of micropropagated medicinal plants.Ther et al. , Sevimayr et al. , and Danr et al. , who emp L. iberica. In conclusion, this study establishes an efficient regeneration protocol for micropropagation of this plant with large potential for use in pharmaceutical industry using young cotyledon node explants. This technique should be a very useful tool for large scale propagation strategies and will serve importantly future Lallemantia agronomists and breeders.This is the first report of plantlet regeneration from cotyledon node explants of


Background: Albuminuria is strongly associated with future risk for cardiovascular and kidney outcomes, and has been proposed to be included in the classification of chronic kidney disease (CKD) along with glomerular filtration rate (GFR). Few data are available on whether albuminuria is associated with concurrent complications of CKD. Methods: A cross-sectional analysis of 1,665 participants screened for the Modification of Diet in Renal Disease (MDRD) study was performed to examine the association between albuminuria ) and measured GFR  with anemia, acidosis, hyperphosphatemia, and hypertension. Results: Mean GFR (± SD) was 39 ml/min/1.73 m2 (± 21) and the median (25 – 75th percentile) ACR was 161 (38 – 680) mg/g. In multivariable models adjusted for age, sex, race, kidney disease etiology, and GFR, higher ACR levels were not associated with any complication. For example, comparing ACR > 300 mg/g vs. < 30 mg/g, the prevalence ratio (95% CI) for anemia was 0.98 (0.81 – 1.20), acidosis 1.13 (0.86 – 1.48), hyperphosphatemia 1.69 (0.91 – 3.17), and hypertension 1.04 (0.97 – 1.12). Lower levels of GFR were associated with all complications. For example, GFR levels < 30 ml/min/1.73 m2 vs. GFR levels 60 – 89 ml/min/1.73 m2 were associated with prevalence ratios (95% CI) of anemia 4.35 (3.18 – 5.96), acidosis 5.31 (3.41 – 8.29), hyperphosphatemia 23.8 (7.71 – 73.6), and hypertension 1.21 (1.10 – 1.32). Conclusions: Albuminuria is not associated with complications after controlling for GFR in patients younger than 70 years of age with non-diabetic CKD and GFR less than 90 ml/min/1.73 m2 and thus would not affect clinical action plans for decisions regarding evaluation and treatment of complications in similar populations.  We cate female] , 23. GFR 125I-iothalamate, as has been previously described [2 [2 were excluded due to the small number of individuals with this level of GFR. Participants with a GFR of 15 – 29 ml/min/1.73 m2 and GFR < 15 ml/min/1.73 m2  were combined into one category due to the small number of participants in Stage 5. Since the entry criteria in the screening phase required creatinine clearance of < 70 ml/min/1.73 m2, GFR category 60 – 89 essentially consisted of patients with GFR 60 – 70 ml/min/1.73 m2 only. GFR was measured using urinary clearance of escribed . Based oOther variables Kidney disease was initially classified into 23 etiologic categories based on history, biopsy, and physician diagnosis but for this analysis it was collapsed into 3 broader categories as previously defined : glomeruThe definitions of anemia, acidosis, and hyperphosphatemia were based on accepted cut-offs for patients with CKD , 26, 27.th – 75th percentiles for skewed variables. The trends across the ACR categories for the characteristics were tested using Spearman rank correlation for continuous variables and Cochran Armitage trend test for categorical variables. We summarized clinical characteristics according to ACR categories using proportions for categorical variables and means and standard deviations (SD) for normally distributed continuous variables or medians and the 25We first explored the optimal functional form for ACR using generalized additive models of log ACR for association of each outcome. Since we did not find any non-linear relationships between albuminuria and any of the complications we used the categories of ACR described above in the primary analysis. 2, as the reference groups. We wanted to express results using prevalence rations as the number of individuals with specific complications was high, and under this circumstance prevalence ratios as obtained by log-binomial regression are preferable. Models were initially unadjusted, but sequential models adjusted for age; age, sex, race, and GFR (or ACR); and age, sex, race, kidney disease etiology, and GFR (or ACR). P-values for linear trend were calculated by modeling category of ACR and GFR as continuous variables in the log-binomial models. We then analyzed the prevalence of the complications across the joint distribution of the ACR categories and GFR categories. We tested the interaction of ACR and GFR categories for modification of the effect of ACR on the complications by level of GFR. We used log-binomial regression to evaluate the association between ACR category and GFR category, separately, to each of the complications, using ACR < 30 mg/g and GFR 60 – 89 ml/min/1.73 mIn sensitivity analyses, we repeated the analyses using proteinuria in place of ACR, categorizing proteinuria by < 200 mg/24 h, 200 – 999 mg/24 h and ≥ 1,000 mg/24 h . We alsoStatistical analyses were performed using SAS (version 9.2). A two-tailed p-value of < 0.05 was considered statistically significant. 2, median (25th – 75th percentiles) for ACR and proteinuria were 161 mg/g (38 – 680) and 320 mg/24 h , respectively. Patients with higher ACR were younger, less likely to be white and male and had lower GFR . A higher proportion of glomerular disease was noted in the higher ACR category. The baseline characteristics of the study population according to the ACR categories are listed in The prevalence of anemia, acidosis, hyperphosphatemia, and hypertension was 43%, 31%, 16%, and 81%, respectively. There was a graded increase in the prevalence of anemia, acidosis, hyperphosphatemia and hypertension with increasing levels of albuminuria . After aThe prevalence of anemia, acidosis, hyperphosphatemia, and hypertension increased with decreasing GFR (p-trend < 0.001 for each complication) . In age-2, but not 30 – 59 or 60 – 89 ml/min/1.73 m2, increasing levels of albuminuria were associated with a higher prevalence of anemia, hyperphosphatemia, and acidosis. However, the interaction between ACR categories and GFR categories was not significant  for each outcome. In sensitivity analysis, the results using proteinuria were similar to those using ACR . For exaIn this study of participants with predominantly non-diabetic CKD, after adjustment for age, race, sex, GFR, and etiology of kidney disease, there was no association between ACR and concurrent complications of CKD, and ACR did not appear to modify the effect of GFR on complications. In contrast, consistent with previous reports, we observed strong associations between lower levels of GFR and each complication studied , 29. The2 and ACR ≥ 10 mg/g were independently associated with all-cause and cardiovascular mortality risk in the general population [Many studies have demonstrated that albuminuria is an independent predictor of mortality, cardiovascular outcomes, and progression of CKD , 30, 31.pulation . In a gepulation . The keypulation , 34, 35.There are several possible mechanisms by which albuminuria could be also associated with CKD complications. First albuminuria has been associated with systemic inflammation, which could contribute to the development of anemia and hypertension , 37. PosThe strengths of this study include the analysis of a large number of individuals from a well-established cohort with moderate-to-severe CKD, availability of measured GFR, detailed ascertainment of confounding variables including CKD etiology, and use of clinically relevant ACR cut-offs that allows for comparability with future studies from other cohorts and thus allow for generalizability of study results to the current era. The study population is restricted mostly to patients with progressive CKD not due to diabetes and therefore is a unique and homogenous population. The cross sectional study design is appropriate as the question being asked refers to point of care in the management of patients with CKD. 2. A significant proportion of patients have CKD based on proteinuria alone. The results will need to be reproduced in other populations such as diabetic kidney disease and other kidney diseases not represented in the MDRD study. There are several limitations to our study. First, ACR was not measured directly but rather derived from 24-hour urine protein excretion using a relationship observed between ACR and 24-hour protein excretion in the IDNT study, a study of diabetic nephropathy, where albumin is the predominant urinary protein. The MDRD Study, consisted of non-diabetic kidney disease and the total urine protein in this population likely contains in addition to albumin, non-albumin proteins such as β-2 microglobulin, retinol binding protein, and other low-molecular weight proteins . Thus, tIn conclusion, ACR does not provide any additional information about CKD complications beyond what is already known with GFR. These findings should be taken into account when considering the inclusion of albuminuria level along with GFR in the development of clinical action plans associated with the proposed new classification of CKD. GFR and albuminuria represent different aspects of kidney disease and management decisions should be based on their individual association with complications and ability to predict adverse outcomes and should be assessed separately for their clinical utility. Research Support: This research is supported by a National Kidney Foundation/Amgen Research Fellowship grant to Dr. Viswanathan. Dr. Inker is supported by a grant K23-DK081017. Dr. Sarnak is supported by K24DK078204. None declared.


P. tenucuilus, T. tyleranus, T. clypeatus, V. speciosa and T. microcarpus of sub-humid and humid agro-ecological zones. Standard analytical techniques following the AOAC were used for proximate and mineral contents determinations. Vitamins determination followed the established standard protocols of the laboratories where the analyses were conducted. Combined use of nutrient concentration and scores were used to compare the level of the contents in the mushroom species. Significant differences (p < 0.05) in nutrient values were demonstrated between and among the mushroom species obtained from the two agro-ecological zones. On dry weight basis, all proximate compositions were high in mushroom species obtained from the humid zone with exception of the total carbohydrates and energy values. Irrespective of the source of the mushrooms, significant amounts were demonstrated in protein, dry matter, ash and total carbohydrates ranging between 11.56–27.42%, 82.34–99.76%, 10.79–16.87%, and 37.12–61.05%, respectively. In comparison with recommended dietary daily intakes, the K, P, Se, Mn, Cu and Fe contents were relatively high with low Ca, Mg, Zn and Na. Thiamin, folic acid, vitamin C, and niacin levels were high but below the recommended FAO references. Considering mushrooms from different agro-ecological zones, significant differences (p < 0.05) were observed in all mushroom species in P except in T. clypeatus, T. tyleranus, T. microcarpus and T. clypeatus in potassium, T. clypeatus and T. microcarpus in Mg. Mushrooms from humid agro-ecological zones had relatively high overall mineral and vitamin supply potential. In conclusion, consumption of these mushrooms should be encouraged in supplementation of the staple food of the poor people. Hence, solving malnutrition problems in children, pregnant mothers, and the immune compromised patients such as the HIV/AIDs.In Uganda, wild mushrooms are mainly collected during the rainy season and valued as a traditionally nutritious food by the rural poor. However, their nutritional attributes have not been adequately studied and documented. Comparative nutrient composition of five wild edible mushroom species was determined, namely:  Lentinus edodes, Pleurotus species and Agaricus bisporus. A high biodiversity of wild mushroom biota is found throughout the tropics, particularly sub-Saharan Africa, with about 300 edible mushroom species  R. Heim, Termitomyces tyleranus (Otieno), Termitomyces clypeatus (Heim), Volvariella speciosa (Fr. ex Fr.) Singer and Polyporus tenucuilus (Beauv.) Fr. Mushrooms were collected during the same rainy season  from humid and sub-humid agro-ecological zones, with their caps curved downwards before shedding off spores. Identification of the mushrooms was done both locally (at source) and macroscopic features of the mushrooms were examined at the College of Agricultural Sciences and Environmental Management, Department of Botany, Makerere University. On-site samples were prepared for DNA assays, whereby health fresh tissues of mushrooms were aseptically removed from the inner part of the caps, placed between clean papers, labeled and wrapped in aluminum foil. The necessary information concerning each mushroom species such as the habitat, substrate and any associations was sent together with the mushrooms for scientific identification at the Key Laboratory of Systematic Mycology and Lichenology, Institute of Microbiology, Chinese Academy of Sciences, China. Molecular identification was done by amplifying and sequencing the Internal Transcribed Spacer (ITS 4 and ITS 5) of the nuclear ribosomal DNA (nrDNA) of the mushrooms. The sequenced DNA fragments were compared with data set obtained from NCBI GenBank. Herbarium specimens were prepared and deposited in the Makerere University Herbarium.Five edible wild mushrooms species were studied: All mushroom species were cleaned, air-dried, ground and transferred into polythene bags, labeled and sealed to prevent any moisture intake and then stored in a refrigerator at 4°C until required for analysis. Cold samples were allowed to attain room temperature before analysis was done.Three grams (3 g) of each ground air-dried mushroom species were analyzed for their proximate contents using AOAC AOAC  method. For determination of the minerals and vitamins, 3 g of each air-dried mushroom sample was and analyzes done in duplicates. Mineral constituents comprising potassium (K), magnesium (Mg), iron (Fe), calcium (Ca), copper (Cu), manganese (Mn) and zinc (Zn) were determined by Atomic Absorption Spectrophotometry , whereas phosphorus (P) and sodium (Na) determination were determined using a flame photometer . For selenium (Se) analysis, digestion was done using an advanced Microwave digestion system  with nitric acid and hydrogen peroxide, and then analyzed using an inductively coupled plasma (ICP) spectrometer . All minerals were expressed in g/100 g dry weight basis, except selenium (µg/100 g).12 determined by vitamin B12 assay SOP No.: 18/1.3 by Ramula. A. of analytical division, Lifescience facility, India. Alpha-tocopherol analysis was carried out according to Delgado-Zamarreño et al.  of two replicates.Data was subjected to analyses of variance (one-way ANOVA) using the Statistical Package for Social Sciences (SPSS) Program 10.1 version. Significance was accepted at the 5% probability level. An independent sample P. tenuiculus in ash, V. speciosa in dry matter and moisture, T. microcarpus in crude lipid, T. tyleranus in crude fibre and T. clypeatus in ash contents. Compared to other mushroom species, T. microcarpus and T. tyleranus, irrespective of their source, had significantly high crude protein, carbohydrates, crude fiber and with low energy values, whereas P. tenuiculus and T. clypeatus exhibited low ash, crude protein, nitrogen and high crude fibre and energy values. Significant differences (p < 0.05) were also observed between mushrooms from the same agro-ecological zones.The proximate composition and energy values of the wild edible mushrooms are shown in Table clypeatus in P, T. clypeatus and T. microcarpus in Mg; T. tyleranus and T. clypeatus in K and Ca in Volvariella speciosa. Variations were also obtained within mushrooms of the same agro-ecological zone. Mineral deficiencies were more pronounced for major but not for trace minerals.Irrespective of the mushroom source, P and K contents were high and low in Mg, Ca and Na Table . ConsideT. clypeatus in Se, Mn and Zn contents. Species mineral scores  indicate deficiency of riboflavin, β-carotene and α-tocopherol, with high folic acid, vitamin C, niacin and thiamin concentrations  and T. microcarpus (humid region) making up to 35.2–54.9% of 50 g of protein/day of Recommended Daily Intake (RDI), which is within the average value of 19–35% dry weight for dried mushrooms . Mushrooms obtained from humid areas had the highest fat content 2.995–3.79%. However, the fat content was within the range of the reported value of wild mushrooms i.e. 1.1–8.1% on dry weight basis . Since the mushroom species examined contained significant amounts of crude fiber, they could be regarded as good sources of dietary fiber for supplementation of some foodstuffs with less fiber such as vegetables, hence utilized as roughage, and mostly its immune-stimulation effects should not be overlooked.Crude fiber is also part of a healthy diet. It was within the reported value of 3–35% fiber on a dry weight basis Breene  and rangs Aletor . Howevers Aletor  recordedEnergy values of the studied wild mushrooms were low and dependant on both mushroom species and the source Table . They vaLike many other organisms, mushrooms bioaccumulate several minerals from their growth media with the help of its greater part, the mycelia. The latter has the highest growth rate, is compressed in nature and is spread over areas of several square metres  was low contributing about 1.8–8.0% of RDI of 400 mg/day. Magnesium was found to be as low as 0.5–1.6 mg/100 g dry weight in ectomycorrhizal mushrooms  than other trace elements, as well as the recommended FAO daily intake (55 µg/100 g). Sanmee et al.  reportedIron content (10.6.6–22.7 g/100 g) was above the RDI (18 mg/day) as well as the upper level intake (40–45 mg/day) , a value within that of bread and cereals (0.68–9 mg/kg) and slightly lower than in eggs, milk and meat (<1 mg/kg); and green vegetables (2 mg/kg) . However, this value is adequate for all age groups, except in pregnancy and lactation which require 1 mg/100 g of Cu , hence, very low for all age groups , contributing 14.7–28.5% of the recommended FAO/WHO  daily inThese mushrooms showed considerable amounts of niacin (2.13–7.31 mg/100 g), providing only 10.7–36.6% of the recommended daily value (20 mg/day). The niacin value is within the RDI for children, but very low for other groups of people , relatively higher than those contained in fruits (0.02–0.07 mg/100 g) and eggs (trace-0.04 mg/100 g) but comparable to vegetables (0.01–0.12 mg/100 g), cereals (0.29–0.33 mg/100 g) FAO  and cultBeta carotene content was low (12.60–17.70 μg/g dry weight) but within the range of other reported ß-carotene values in wild mushrooms (2.52–75.48 µg/g dry weight, Barros et al. 12 and pantothenic acid, which observation is in disagreement with other reports elsewhere (Breene In addition to ß-carotene, these mushrooms were found to contain trace amounts of α-tocopherol ranging from 0.39 to 0.52 μg/100 g dry weight. Alpha tocopherol is the most active component of the vitamin E complex and a powerful antioxidant of the human body (Burton and Ingold e Breene .Generally, the five mushroom species studied irrespective of their sources are good sources nutrients and compare favorably with the most common green leafy vegetables, legumes, whole grain cereals, meats and dairy products. In Uganda and indeed in other developing countries, micronutrient deficiency is the main cause of death and poor performance due to the poor staple foods consumed. This affects mainly the children, women, HIV-AIDs patients and the elderly. Therefore, if these mushrooms are encouraged to be consumed together with the common staple foods, especially of a poor man’s diet, stand a high chance of solving malnutrition in the above mentioned vulnerable groups of people.


Vanda roxburghii Rbr. root has been used traditionally in Bangladesh as tonic to brain and in the treatment of nervous system disorders including AD. Therefore, we aimed to investigate the cholinesterase inhibitory activities and antioxidant properties of the extracts from V. roxburghii.Alzheimer’s disease (AD) is a progressively developing neurodegenerative disorder of the brain in the elderly people. The crude methanol extract from the roots of plant was sequentially fractionated with petroleum ether, chloroform, ethylacetate and water to yield their corresponding extracts. The extracts were assessed for acetylcholinesterase and butyrylcholinesterase inhibitory activity by modified Ellman method and antioxidant property by several assays including ferric reducing antioxidant power, scavenging of 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical and hydroxyl radical, and inhibition of lipid peroxidation. Endogenous substances in the extracts were analyzed by the standard phytochemical methods and active compound was isolated by the chromatographic methods.50 values of 221.13 and 82.51 μg/ml, respectively. Phytochemical screening revealed a large amount of phenolics and flavonoids in the chloroform extract. Bioactivity guided separation techniques led to the isolation of a strong antioxidant from the chloroform extract and its structure was determined as gigantol on the basis of spectral studies.Chloroform extract was shown to demonstrate strong ferric-reducing antioxidant power and scavenging activity against DPPH and hydroxyl free radicals when compared with the other extracts and the reference standard catechin. The antioxidant effect was further verified by inhibition of lipid peroxidation in rat brain homogenates. Likewise, the chloroform extract exhibited the highest inhibition against both the acetylcholinesterase and butyrylcholinesterase enzymes with ICV. roxburghii, possibly due to its phenolic compounds, exert potential antioxidant and cholinesterase inhibitory activities, which may be useful in the treatment of AD.These results suggest that the chloroform extract of  Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder in the elderly people with symptoms of memory loss, cognition and behavioral abnormalities. The major neuropathological characteristics observed in AD are senile plaque consisting of Abeta protein, and neurofibrillary tangles of microtubule associated protein tau, and loss of cholinergic neurons –4. Currein vivo and in vitro , and centrifuged at 10,000 g for 30 min. The resulting supernatant was used as an enzyme source. For the BChE enzyme source, human blood from anonymous healthy men subject (25 years) was provided by the Rajshahi Univresity Medical Center, Rajshahi, Bagladesh and collected in EDTA treated (1 mg/ml) glass tubes. The tubes were centrifuged at 2000 g for 10 min to eliminate the red blood cells. The resulting plasma (supernatant) was then recuparated, diluted (1/200) with 50 mM phosphate buffer (pH 7.4) and was used immediately for studying butyrylcholinesterase activity.The acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory assay were performed according to the colorimetric method of Ellman et al. . For theThe rates of hydrolysis by acetylcholinesterase were monitored spectrophotometrically. Each extract or standard (500 μl) was mixed with an enzyme solution (200 μl) and incubated at 37 °C for 15 min. Absorbance at 405 nm was read immediately after adding an Ellman’s reaction mixture  in a 50 mM sodium phosphate buffer (pH 8.0) to the above reaction mixture. Reading was repeated for 10 min at 2 min intervals to verify that the reaction occurred linearly. The blank reaction was measured by substituting saline for the enzyme. Donepezil was used as positive control. The percentage inhibition of acetylcholinesterase activity was calculated using the following formula:Assessment of BChE inhibition was performed as described above except that the enzyme solution was 50 μl and acetylthiocholine iodide (ACh) was replaced by butyrylthiocholine iodide (BCh). Galantamine was used as positive control. The percentage inhibition of butyrylcholinesterase activity was calculated using the same formula as mentioned above for acetylcholinesterase activity.V. roxburghii was fractionated by silica gel 60  column chromatography using n-hexane, chloroform and methanol gradient system with gradually increasing polarity. Fractions of 25 ml were collected and concentrated. All eluted fractions were monitored by thin layer chromatography (TLC) and visualized under UV light (254 nm). Fractions with antioxidant activity caused by the same compounds  were combined. A total of five fractions  were obtained. The most active fraction CLC1 (1.1 g) was further separated by preparative thin layer chromatography using n-hexane-acetone (6:4) as the mobile phase to yield compound 1 (14 mg).The chloroform extract (6.0 g) of 1H and 13 C-NMR spectra in CDCL3. The structure of the compound was confirmed by comparison of its spectral data with those published in the literature [Spectral analysis for the compound 1 was achieved by using Jeol-Ex 400 MHz and FT-NMR spectrometers for terature .max, 226 and 282 nm. 1H-NMR (CDCl3): δH 6.81 , 6.68 , 6.62 , 6.60 , 6.24 , 6.29 , 2.82 , 2.79 , 3.82  and 3.74 . 13C-NMR (CDCl3): δC 160.90 (C-5′), 156.67 (C-3′), 146.30 (C-5″), 144.55 (C-1′), 143.81 (C-4″), 133.64 (C-1″), 121.02 (C-2″), 114.32 (C-3″), 111.21 (C-6″), 108.07 (C-6′), 106.83 (C-4′), 99.06 (C-2′), 55.90 (C5″-OCH3),, 55.27 (C5′-OCH3), 38.29 (C-2), and 37.26 (C-1).Gigantol (1): colorless gum, UV (in MeOH) λ± SD. Free R-software version 2.15.1 (http://www.r-project.org/) and Microsoft Excel 2007  were used for the statistical and graphical evaluations. Significant differences  between the means were determined using the t-test.All analyses were carried out in triplicates. Data were presented as mean V. roxburghii, an epiphyte has been used in Unani traditional medicine as tonic to brain and liver and also used by the local people of Bangladesh to treat nervous system disorders [V. roxburghii for cholinesterase inhibitory and antioxidant activities.The practice of traditional medicine has described a wide range of plants and herbs which may be used in the treatment of AD. Such plants can play important roles in providing alternative medicine, adjuncts to existing therapies, and in the discovery of new pharmacological compounds. Indeed, one of the most widely used anti-AD drug, galantamine is derived from the plant . V. roxbisorders –26. In tV. roxburghii viz. petroleum ether, chloroform, ethylacetate and aqueous extracts were analyzed qualitatively for the presence or absence of phytochemicals. The extracts showed the presence of tannins, phenolics and flavonoids, phytosterols, alkaloids and saponin  and DPPH radical scavenging activity. Both assays demonstrated the potential antioxidant activity of the chloroform extract which was close to the activity of the reference standard catechin  and prostaglandin E2 (PGE-2) production in RAW 264.7 macrophage cells. The compound is also reported to reduce carrageenan-induced inflammation in rats [Gigantol, a bibenzyle compound has been found to be an important constituent in the orchid plants and studied for a number of biological activities. Simmler et al. (2011) showed that gigantol from oduction . In a production , giganto in rats . Several in rats . Since gV. roxburghii, possibly due to its polyphenolic compounds, possess a combination of antioxidant properties and cholinesterase inhibitory activities which support its traditional utilization in Bangladesh in the treatment of AD. In a reference survey, no reports relating to the antioxidant and cholinesterase inhibitory activities of V. roxburghii has extracts have been found so far. Besides these, gigantol, a bibenzyl stilbenoid was isolated from the chloroform extract as the potent antioxidant principle.Results of the present study clearly demonstrated that the chloroform extract of


Objective. To evaluate the effects of ezetimibe/simvastatin (EZE/SIMV) and rosuvastatin (ROSUV) on oxidative stress (OS) markers in patients with diabetic polyneuropathy (DPN). Methods. We performed a randomized, double-blind, placebo-controlled phase III clinical trial in adult patients with Type 2 Diabetes Mellitus (T2DM) and DPN, as evaluated by composite scores and nerve conduction studies (NCS). Seventy-four subjects with T2DM were allocated 1 : 1 : 1 to placebo, EZE/SIMV 10/20 mg, or ROSUV 20 mg for 16 weeks. All patients were assessed before and after treatment: primary outcomes were lipid peroxidation (LPO), and nitric oxide (NO) surrogate levels in plasma; secondary outcomes included NCS, neuropathic symptom scores, and metabolic parameters. Data were expressed as mean ± SD or SEM, frequencies, and percentages; we used nonparametric analysis. Results. LPO levels were reduced in both statin arms after 16 weeks of treatment (p < 0.05 versus baseline), without changes in the placebo group. NO levels were not significantly affected by statin treatment, although a trend towards significance concerning increased NO levels was noted in both statin arms. No significant changes were observed for the NCS or composite scores. Discussion. EZE/SIMV and ROSUV are superior to placebo in reducing LPO in subjects with T2DM suffering from polyneuropathy. This trial is registered with NCT02129231. Nerve damage in patients with diabetes is known as diabetic neuropathy and is considered as the most prevalent microvascular complication—up to 60%—in Type 2 Diabetes Mellitus (T2DM) subjects . DiabetiThe 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are potent inhibitors of cholesterol biosynthesis. Various clinical trials have shown beneficial effects of statins in the prevention of cardiovascular diseases . However in vitro [Simvastatin (SIMV), a lipophilic statin with intrinsic antioxidant activity, has been demonstrated to possess higher antihydroxyl radical activity than other statins . Similarin vitro , as wellin vitro .Thus, based on the aforementioned evidence regarding the plausible relationship between OS and DPN, and the promising pleiotropic effects of statins on this scenario, this randomized clinical trial was performed to evaluate the value of SIMV and ROSUV concerning the reduction of OS in patients with T2DM and DPN.A randomized, double-blind, placebo-controlled phase III clinical trial was performed at the Clinical and Experimental Therapeutics Institute, University of Guadalajara, Mexico. Subjects were assigned to three group treatments in blocks with a parallel sequence 1 : 1 : 1, through a randomized computer-based list generated by a different researcher unaware of the drugs given. Patients received once-a-day single-dose for 16 weeks of each treatment: controls received placebo, ezetimibe/SIMV (EZE/SIMV) 10/20 mg, and ROSUV 20 mg. We wanted to evaluate two high potency statins; in our Country, we lack rosuvastatin 5 mg dosage. Simvastatin monotherapy doses of 80 mg/day equal rosuvastatin 10 mg; however, high doses of statins increase the risk of adverse reactions; that is why we chose a combination therapy to reach similar effects and reduce adverse events. Patients were instructed to take their drugs in the evening at the same time every day. All drugs were similar in physical characteristics and presented in dark bottles, carefully filled by another group researcher who placed a respective tag with the patient code. Apart, patients were provided with a diary, whereby they wrote down the date and time of drug administration, as well as any drug adverse reactions experienced. Compliance was assessed by a coresearcher through pill counting and review of the diary provided. Such information was collected and registered every 4 weeks. The selection period was performed from February 2012 to January 2013. We did not influence or change their standard medications or lifestyle  during the study. Their family doctor was in charge of ensuring metabolic control, and we established frequent communication with them, to ensure none of the drugs implemented in our protocol suffered modifications. Also, patients were referred to their family physician or specialist if urgent treatment with statins or vitamin supplementation was required.Inclusion criteria were as follows: ≥18 years old, T2DM defined by the American Diabetes Association criteria, DPN defined by Dyck  criteriaμM.When polyunsaturated fatty acids are oxidized by ROS, malondialdehyde (MDA) is produced upon fatty acid decomposition; thus, measurement of MDA has been used as an indicator of LPO. LPO plasma levels were measured by a commercial kit  according to manufacturer's instructions. This assay is based on the reaction of a chromogenic reagent, N-methyl-2-phenylindole, with MDA, which produces a chromophore with maximal absorbance at 586 nm. Results are expressed in x), according to manufacturer's instructions using a colorimetric assay kit . Plasma NOx quantification is based on the Griess reaction. In brief, nitrate is converted to nitrite with cofactor and nitrate reductase, and then total nitrite reacts with the Griess reagent, thereby forming a deep purple azo compound which absorbs light at 540 nm. Results are expressed as pmol/mL.The plasma NO levels were indirectly estimated based on the determination of the NO metabolites, nitrate, and nitrite .The study was approved by the Research and Ethics Committee of the Health Science University Center, University of Guadalajara, Mexico, and by international instances  with clinical trial identifier μM in LPO, 95% confidence interval, 80% potency, and two-tailed p < 0.05, which resulted in 21 for each group. Quantitative variables were expressed as mean ± SD or SEM. Kolmogorov-Smirnov and Shapiro-Wilk tests were performed to determine the distribution of variables. Friedman and Wilcoxon tests were used before and after analysis, and Kruskal-Wallis with Mann-Whitney U as post hoc analysis between groups comparison. Qualitative variables were expressed as frequencies and percentages. McNemar test was used to evaluate differences in dichotomy variables before and after treatment and between groups comparison Fisher's exact and χ2 tests were used as appropriate. Significance level was established with a p value <0.05.The sample size was obtained by a clinical study design formula taking into account a difference change of 0.05 We assessed 131 patients, 57 were not eligible, and 74 were included and further divided into groups as follows: placebo, 24; EZE/SIMV, 25; and ROSUV, 25 . There wμM, respectively . After 16 weeks, placebo group showed increased LPO levels up to 1.31 ± 0.20 μM   and 0.53 ± 0.10 μM , respectively  . On the ectively .p = 0.15, Kruskal-Wallis). After 16 weeks, placebo group showed 120.25 ± 41.8 pmol/mL , EZE/SIMV 168.57 ± 54.13 pmol/mL , and ROSUV 211.73 ± 75.05 pmol/mL  NO levels.Basal NO levels in the placebo, EZE/SIMV, and ROSUV groups were 156.0 ± 47.58, 83.43 ± 31.72, and 108.3 ± 30.36 pmol/mL, respectively . At basep = 0.851 between groups), respectively. We observed significant reductions in the placebo group to 2.2 ± 1.6 , EZE/SIMV 1.4 ± 1.5 , and ROSUV 1.5 ± 1.4 . Noteworthy, the EZE/SIMV and ROSUV groups showed greater improvement than placebo group (p = 0.110 between groups) , EZE/SIMV , and ROSUV  groups. Basal analogue pain scale (APS) values were 5.2 ± 0.7, 4.4 ± 0.8, and 3.9 ± 0.7 (p = 0.463 between groups), in the placebo, EZE/SIMV, and ROSUV groups, respectively. We observed a reduction on APS values of 2.6 ± 0.0 , 2.1 ± 0.0 , and 1.5 ± 0.1  in the placebo, EZE/SIMV, and ROSUV groups, respectively  . At the ectively .p < 0.01 baseline versus final) and an increase of 1.3 and 1.0 m/s on the peroneal and tibiae nerve velocities, respectively . In the EZE/SIMV group there were a reduction of 0.4 ms on the sural nerve latency , an increase of 1.5 mV on the median motor nerve amplitude , and an increase of 0.4 mV on the median sensitive nerve latency .The electrophysiological data are shown in p = 0.013), HbA1c (p = 0.044), and total bilirubin (p = 0.003). At the end of the study, there was a reduction in fasting plasma glucose in the placebo group (p = 0.004) and LDL (p = 0.01). In the EZE/SIMV group there were a significant reduction on TC 82.8 ± 49.8 mg/dL (p = 0.001) and LDL 57.1 ± 48.4 mg/dL (p = 0.001) and a trend towards significance on TG levels (p = 0.055). In the ROSUV arm CT, LDL, and TG were reduced by 73.3 ± 49.5 mg/dL (p = 0.001), 64.9 ± 44.0 mg/dL (p = 0.001), and 41.1 ± 61.2 mg/dL (p = 0.003), respectively. Also, a significant reduction was observed concerning bilirubin levels (p = 0.02). We also report on gastrointestinal, neurologic, dermatologic, and muscular adverse drug reactions, and 2 patients were eliminated due to statin-related myopathy (one in each group) (see Metabolic characteristics are shown in oup) see .Diabetes can damage the peripheral nervous system in various ways, DPN being the most common presentation . DPN is There are several underlying mechanisms suggested to be linked to the development and progression of DPN caused by dyslipidemias . A conveFurthermore, as patients with diabetes exhibit impaired NO availability, thereby contributing to endothelial dysfunction , we evalp = 0.052), which may suggest that a lengthened treatment is necessary to achieve a significant difference. As expected, the lipid profile was favorably affected  by the statin treatment when compared to placebo. Unexpectedly, the placebo group improved fasting plasma glucose, without repercussion on HBA1C. Regarding safety issues, we did not observe any significant elevation neither on liver enzymes nor creatinine by treatment with statins.Although OS has been implicated in the development and progression of diabetic neuropathies, we did not observe a superior effect of statins over placebo on the clinical outcomes after 16 weeks of intervention. However, we observed a trend towards significance on the NSS values in the EZE/SIMV group when compared to placebo (Limitations include the lack of homogenization of lifestyle changes previous to randomization and throughout the study. Some of the patients could change their antidiabetic medications during the protocol, because their family physician was in charge of their glucose control; however, we ensured no statins and/or antioxidants were taken during the duration of the study. Most of the clinical trials that evaluate DPN are performed for a minimum of 12 months to ensure modifications in clinical outcomes; probably the duration of our trial was too short to demonstrate changes in clinical and nerve conduction parameters; however, the main objective was to evaluate the statins effect on oxidative stress.In summary, this trial demonstrated that EZE/SIMV and ROSUV are superior to placebo in reducing LPO levels in T2DM after 16 weeks of treatment. Future larger randomized clinical trials and for longer period of time are needed, in order to confirm the favorable effects that statins may have on OS in T2DM subjects suffering from DPN.


The aim of this study was to investigate the maintenance of volume as a spacer by comparing vascular supply and apoptosis in an implanted autologous-free dermal fat graft (FDFG) and free fat graft (FFG). An autologous FDFG is a material used in plastic surgery and oncoplastic breast surgery that is ideal for immediate volume replacement after partial mastectomy because of its easy availability and minimal invasion of the donor site; however, immunohistochemical findings and survival procedures have not yet been reported.An experimental protocol using a unique animal model was designed for the present study. The expression of vascular endothelial growth factor (VEGF) was measured in FDFGs and FFGs implanted onto the pectoral major muscle of Wistar rats. Thirty Wistar rats were divided into two groups and postoperatively 1, 2, 4, 8, and 16 weeks . Six samples from three rats in each group were used as control samples (POW0).The thickness of the implanted FDFG was not significantly different from the control sample at POW1, 2, 4, 8, and 16 between FDFG and FFG group; however, the thickness at POW8 and 16 was significantly lesser in the FFG group than in the control samples.The average proportion of fatty tissue to whole tissue ranged from 34.2 to 48.6 % in the FDFG group and from 57.2 to 76.7 % in the FFG group during the observation period; however, there was no significant difference in the proportion of fatty tissue between these two groups. There were no significant differences between the average number of VEGF-positive cells in the FDFG group and the FFG group at POW1, 2, 4, 8, and 16. The average number of TUNEL-positive cells in the early period at POW1 was significantly lower in the FDFG group than in the FFG group.This rat model was useful for investigating the mechanisms of angiogenesis, apoptosis, structure maintenance, and fibromatous changes. From the present experimental study, we believe that FDFG is one of the most convenient materials currently available to repair small defects at the time of BCS even in the clinical field. Oncoplastic breast surgery, in combination with oncological and plastic procedures during surgical treatment for early breast cancer, has become popular, especially in Western countries –3. ThereAlthough several studies have reported transplantation using free fat grafts (FFGs) or fat injections, we could not find any literature regarding an investigation of FDFG. The purpose of this study was to histologically evaluate FDFG implanted onto the major pectoral muscle to evaluate the condition of fatty cells in animal models, and to predict the role of oncoplastic surgery using FDFG at the time of BCS.Thirty-six 7-week-old male Wister rats weighting 200 g were bred in wire cages until their weight increased to 600 g (640–810 g). Thereafter, we could easily perform autologous implantation of FDFG and a FFG.After 20-week-old rats were anesthetized by inhaling diethyl ether, incision areas were marked on the lateral abdomen Fig. . To harvThree rats implanted with FDFGs and FFGs were euthanized 1, 2, 4, 8, and 16 weeks postoperatively, respectively . The implanted FDFG/FFGs with the pectoralis major muscle and skin were resected in a lump as one block. Six samples, two samples from one rat, were collected for each postoperative period in the FDFG and FFG groups. After being fixed immediately in neutral buffer formalin for 48 h, they were embedded in paraffin. Samples were cut at a thickness of 3 μm and were fixed onto glass slides retreated with 3-aminopropyltriethoxysilane . Six FDFG samples and six FFG samples were collected as control samples, respectively. There were immediately fixed in neutral buffer formalin for histological evaluation.A total of 6 samples in the FDFG and FFG groups 1, 2, 4, 8, 16 weeks postoperatively were analyzed using hematoxylin and eosin (HE) and immunohistochemical staining. Six samples obtained at POW0 from the two groups were used as control samples. One representative section in the largest portion of each FDFG or FFG sample was selected for histological examination.At 40× objective on HE staining, we microscopically measured the thickness of FDFG or FFG as a whole. At 100× objective, 5 fields were randomly selected from HE-stained sections, and fatty tissue areas were measured using ImageJ  . The proTo evaluate microvascular angiogenesis and the apoptosis of adipose cells on each dimension of FDFG/FFG, we selected 5 fields including a central area, a pectoralis major muscle side, a skin side, and the lateral sides of FDFG/FFG for immunohistochemical staining. Representative sections were stained using a streptavidin–biotin–peroxidase supersensitive kit , with a rat monoclonal anti-VEGF antibody  for angiogenesis at a concentration of 1:100. The endogenous peroxidase activity of specimens was blocked by immersing the slides in a 3 % hydrogen peroxidase-methanol solution for 30 min at room temperature. After washing three times with PBS for 5 min each, sections were treated with 1 % bovine serum albumin for 30 min to block non-specific reactions at room temperature. Then, sections were incubated with the primary antibody to VEGF (1:100) at 4 °C overnight and were subsequently stained with secondary antibodies for 30 min. Sections were incubated with the avidin–biotin complex for 60 min and reactions were visualized using diaminobenzidine tetrahydrochloride for 2 min. Sections were rinsed briefly in water and counterstained with hematoxylin for 30 s. An area was selected randomly to evaluate the expression of VEGF on each section and measure the contribution of VEGF-positive cells by ImageJ . TUNEL staining  was used to detect apoptotic cells –10. Posit test. A p value of <0.05 was considered significant. Data were expressed as the mean ± SD.We compared the thickness of each postoperative period, and the expression of VEGF-positive cells and TUNEL-positive cells between the two groups using a two-sided Student’s A total of 72 samples from 36 rats were collected and evaluated as follows: 18 rats were POW0, 1, 2, 4, 8, and 16 in the FDFG group, 18 were POW0, 1, 2, 4, 8, and 16 in the FFG group. No necrotic changes or infections were observed. On HE staining, fatty tissue and eosinophilic fibrous tissue were recognized separately , 9.8 mm 4.8–14.0), 9.3 mm (5.0–12.5), 4.5 mm (3.5–6.0), and 3.7 mm (1.5–6.5) in the FDFG group at POW1, 2, 4, 8, 12, and 16, respectively. The average thickness was 6.8 mm (5.1–8.0), 6.8 mm (5.8–8.2), 4.1 mm (3.4–4.5), 2.4 mm (1.4–2.9), 1.8 mm (1.1–2.5) in the FFG group at POW1, 2, 4, 8, and 16, respectively , 51.2 % (10.4–71.5), 48.6 % (30.0–63.3), 45.0 % (14.7–69.4), and 34.2 % (4.2–74.3) in the FDFG group, and 76.0 % (63.3–80.9), 76.7 % (69.7–83.4), 73.1 % (69.9–75.3), 71.8 % (61.1–77.3), and 57.2 % (48.6–64.1) in the FFG group at POW1, 2, 4, 8, and 16, respectively. Also, there was no significant difference between each postoperative value and the control value in both groups. At each period, there was no significant difference in the proportion of fatty tissue between the FDFG and FFG groups , 45258.8 pixels (18822.0–62806.0) (p < 0.0001), 32784.2 pixels (18194.3–48038.7) (p < 0.0001), 34009.5 pixels (24508.7–53673.3) (p < 0.0001), and 32821.9 pixels (8779.7–54988.3) (p = 0.0019) at POW1, 2, 4, 8, and 16, respectively.VEGF-positive cells were observed in all sections. They could be seen in the extracellular matrix around adipose cells Fig. . The avep = 0.1120), 601.8 pixels (171.0–1528.7) (p = 0.8027), 770.9 pixels (651.3–1194.7) (p = 0.2656), 786.3 pixels (441.7–1615.3) (p = 0.3140), and 752.3 pixels (622.0–857.3) (p = 0.2212) at POW1, 2, 4, 8, and 16, respectively. VEGF-positive cells were higher in the FDFG group than in the control group at all periods. The average number of VEGF-positive cells was significantly higher in the FDFG group than in the FFG group at POW1, 2, 4, 8, and 16  . The proportion of fatty tissue was slightly lower in the FDFG group than in the FFG group although it was not significant. From pathological findings in the present study, the larger thickness in size of FDFG group depends on the volume of parenchymal tissue, such as fibrosis, infiltration or of lymphocyte except fatty tissue. Attaching dermis to fatty tissue may influence something to the parenchymal tissue resulting in keeping better volume than that without any dermis. If the greater volume of FDFG group depends on the volume of parenchymal tissue, the results of TUNNEL method might show the positive relationship between apoptotic cells and parenchymal volume of implanted FDFG/FFG. The fact that thickness of the grafts was measured in histologic sections implies that the shrinkage and other eventual changes which occur with formalin fixation and the various tissues dehydration steps would have made these results inaccurate from “real state”. We did not measure the thickness at the time of explanation. To avoid producing gaps between the grafts and surrounding tissue, such as pectoral muscle, fatty tissue, and skin, we did not make the direct intervention before an adequate fixation. However, we should have been able to make another experimental rat model for the right measurement of the fresh sample but not for histological examinations. The following examination may be needed.In this study, we developed a rat model implanted with FDFGs and FFGs. We showed that the histological findings of implanted FDFGs on the pectoralis major muscle resembled the clinical results we previously reported on FDFGs implanted onto partial breast defects with early breast cancer. These findings were not found in other studies. We also hypothesized from clinical experience that implanted FDFGs may undergo mild resorption and degeneration to fibrous tissue, but that the volume they filled may be maintained long after surgery as a “spacer”. In the present study, the thickness of the implanted graft gradually decreased, but was less affected in the FDFG group than in the FFG group (Higher expression of VEGF and lower apoptosis was measured in the FDFG group than in the FFG group. These results suggest that FDFG may maintain volume at least until the end of the observation. Although the proportion of fatty tissue was slightly lower in the FDFG group than in the FFG group (without a significant difference), the total volume of implanted tissue was larger in the FDFG group. Various factors for angiogenesis, such as the VEGF family, have been reported to play an important role in the revascularization of the graft . In the Our new rat model in which FDFG was implanted onto the pectoralis major muscle was very useful for investigating the mechanisms of angiogenesis, apoptosis, structure maintenance, and fibromatous changes. From the present experimental study, we believe that FDFG is one of the most convenient materials to repair small defects at the time of BCS.We histologically investigated the role of attaching a dermis to FFGs in an autologous transplantation from the lateral abdomen onto the major pectoral muscle in a rat model. FDFG maintained blood flow and apoptotic resistance.


Statistical analysis demonstrated the following for flexural strength (P < 0.05): I-sil: 155.89 ± 45.27BC; P-sil: 155.89 ± 45.27BC; I-sil/heat: 130.20 ± 22.11C; P-sil/heat: 169.86 ± 50.29AB; I: 131.87 ± 15.86C. For compressive strength, the following are demonstrated: I-sil: 1367.25 ± 188.77ab; P-sil: 867.61 ± 102.76d; I-sil/heat: 1162.98 ± 222.07c; P-sil/heat: 1499.35 ± 339.06a; and I: 1245.78 ± 211.16bc. Due to the impossibility of incorporating the stipulated amount of fiber, P group was excluded. Glass fiber treatment with heated silane enhanced flexural and compressive strength of a reinforced dental methacrylate.This study evaluated the influence of silane heat treatment and glass fiber fabrication type, industrially treated (I) or pure (P), on flexural and compressive strength of methacrylate resin bars . Six groups ( Fiber reinforced composites properties can be manipulated according to the required use, creating the possibility of changes in position, orientation, and quantity of inserted fibers. Engineering sciences have developed composites with inclusion of strong fibers within a binder matrix ; glass fFiber reinforcement of restorative dental materials –12 and eIn 2006, Garoushi et al.  evaluateIn order to improve the adhesion between polymer and glass fibers, a silane coupling agent has been used for several decades . AdhesioStudies have shown that heat treatment of silanated surfaces at 100°C consolidates the condensation reaction and increases their bonding strength through the elimination of water, alcohol, and other subproducts of the condensation reaction . HoweverThe aim of this study was to evaluate the influence of silane heat treatment and the short glass fiber fabrication type  on compressive and flexural strength of methacrylate resin bars.N = 10) were created according to the interaction of factors in study (2 × 2) plus 2 control groups . Pure fibers were cut to the same length. In I-sil, P-sil, I-sil/heat, and P-sil/heat groups, fibers received surface treatment with silane coupling agent. After the silanization, the fibers of I-sil/heat and P-sil/heat groups were heat treated at 100°C by an air blow dryer for one minute and then manually incorporated into the resin according to the experimental groups.n = 10) were stored in distilled water at 37°C for 24 hours before testing. A compressive load was applied at the center of the specimens to obtain the compressive strength at a crosshead speed of 0.5 mm/min, in a universal testing machine , and the maximum load to break was recorded in N.A condensation silicon impression material  mold was made from a stainless pattern to produce standardized rectangular specimens with dimensions of 9.5 mm × 5.5 mm × 3 mm, according to Garoushi et al. . The EC A condensation silicon impression material  mold was made from a stainless pattern to produce standardized rectangular specimens with dimension of 10 mm × 2 mm × 1 mm, according to Pick et al. . The EC PL/wb2, where “P” is the maximum load at fracture, “L” is the distance between the supports (20 mm), “w” is the sample thickness, and “b” is the height. The samples' thickness and height were measured with a digital caliper .The specimens were stored in distilled water at 37°C for 24 hours prior to test, and after that they were positioned on a 3-point bending flexural strength testing apparatus (Instron 5965) with two supports 20 mm apart and tested at a crosshead speed of 0.5 mm/min. The flexure strength (FS) was calculated in MPa with the following equation: FS = 2A factorial analysis was performed on a 2 × 2 design with a general linear model procedure, excluding control groups and comparing factors in study: silane coupling agent application, in two levels (heated or not heated), and short glass fiber fabrication type, in two levels . After that, data were first submitted to Kolmogorov-Smirnov test to verify the normality of distribution and subsequently analyzed by ANOVA and Tukey-HSD test for comparisons among groups. All tests were performed at a significance level of 5% in SPSS 20.0 software .P = 0.008) or for compressive strength tests (P = 0.0001). ANOVA, following Tukey-HSD test, results for flexural and compressive strength are shown in The factorial analysis showed significant interaction between factors in study, either for flexural strength  negatively reacts with heated silane application. Based on this fact, it is suggested that such treatment on these fibers affects the fiber-to-resin adhesion when subjected to heat treatment, generating low values of flexural and compressive strength.The major limitation of this study was the difficulty of manipulation of the experimental resin due to the absence of filler particles. The created experimental composite had high fluidity, being difficult to handle. Further studies are needed to improve the experimental properties of this composite. Also the increase of number of specimens per group could have returned a more significant result, reducing standard deviation; in spite of that, all tests showed significant results, which support the following conclusions.Within the limitations of this study, it is possible to conclude that the heat treatment of the silane coupling agent is significant to increase the flexural and compressive strength of a dental methacrylate polymer reinforced with pure short glass fibers.


Participants exercised for 60 min or until volitional fatigue, core temperature reached 39°C, or heart rate exceeded 90% of maximum. Tolerance time, core temperature, skin temperature, mean body temperature, heart rate, and body mass loss were measured. Exercise time was reduced in the higher WBGT environments  and work intensities . The majority of trials  were terminated due to participant's heart rate exceeding 90% of their maximum. A total of eight trials (7.4%) lasted the full duration. Only nine (8.3%) trials were terminated due to volitional fatigue and six (5.6%) due to core temperatures in excess of 39°C. These results demonstrate that physiological tolerance times are influenced by the external environment and workload and that cardiovascular strain is the limiting factor to work tolerance when wearing this heavy multilayered PPE.This study evaluated the physiological tolerance times when wearing explosive and chemical (>35 kg) personal protective equipment (PPE) in simulated environmental extremes across a range of differing work intensities. Twelve healthy males undertook nine trials which involved walking on a treadmill at 2.5, 4, and 5.5 km·h Personal protective equipment (PPE) is required in sporting and occupational settings to protect the wearer from a range of hazards . UnfortuSeveral studies have examined the physiological strain encountered by fire fighters , police We have previously examined the physiological effects of wearing EOD PPE in the field , 18 and Thus, the purpose of the present investigation was to examine the physiological tolerance times while wearing chemical and explosive protective clothing concurrently across a range metabolic work rates and ambient environments. Establishing this information has implications for determining safe tolerance times for EOD technicians when required to wear this PPE in various environments.2, sum of eight skinfolds 79.1 ± 31.6 mm, maximal oxygen uptake : age = 24.1 ± 3 years, height = 1.79 ± 0.06 m, body mass = 76.4 ± 8.4 kg, body surface area 2.0 ± 0.1 mParticipants attended the laboratory on four separate occasions, at the same time of day, separated by a minimum of 7 days. In the initial visit height and nude body mass were recorded and body surface area was subsequently calculated . Skinfolreadmill . ParticiIn the three remaining laboratory visits participants wore a fully encapsulating NFPA 1994 Class 3 chemical/biological protective garment, including outer gloves and booties , and a respirator . The garment was made from trilaminate, a three-layer chemical/biological protective fabric, consisting of a selectively permeable barrier film laminated between outer and inner textiles. A Med-Eng EOD9 suit  consisting of a jacket, trousers, groin protection, and a helmet (33.4 kg) was donned over the chemical PPE and respirator. The combined weight of the ensemble was 35.45 kg. Participants' base layer consisted of a t-shirt, shorts, socks, and underwear . Athleti−1 and a radiant heat load (two radiant heaters positioned ~1.3 m from the participant) were incorporated throughout the testing. These environmental conditions were also monitored independently . During each of these laboratory visits the participant completed three treadmill-walking trials of 2.5, 4, and 5.5 km·h−1 with a 1% gradient. This equated to an external work rate [−2 for a 76 kg individual with a body surface area of 2.0 m−2. The order of the testing, for both the work rate and the environment, was randomised using a random number generator in a controlled crossover design.During the trials the participants walked on a treadmill, while wearing the PPE, in an environmental chamber . A wet bulb globe temperature (WBGT) of 21, 30, or 37°C was obtained by the following ambient temperatures and relative humidities: 24°C, 50%; 32°C, 60%; and 48°C, 20%; respectively. A simulated wind speed equivalent to ~4.5 km·hork rate  of ~135,During each trial, standard termination criteria were applied in accordance with the American Society for Testing and Materials guidelines : (1) corThe primary outcome measure of the current study was physiological tolerance times measured to the nearest 0.5 min. Core temperature was recorded using an ingestible sensor  swallowed ~6 hours before each trial. This was to allow sufficient time for the sensor to pass from the stomach to the intestines, where the reading of core body temperature is optimal . WeightePretrial hydration status was confirmed using urine specific gravity  of <1.020. If participants did not meet the above guidelines they were given an additional 500 mL of water to be consumed prior to commencement of the trial. Nude body mass was undertaken following towel drying and measured to the nearest 50 g  before and after each trial. A cannula was inserted in the antecubital fossa for the attainment of venous blood samples in five mL serum separating vacutainers for the determination of serum osmolality using the freezing point depression technique  as previously described , 34. SerF-ratio. Tolerance times, body mass loss, and the final value recorded for core temperature, skin temperature, mean body temperature, heart rate, and PSI were analysed using a two-way (environment × work intensity) repeated measures analysis of variance (ANOVA). To determine if baseline physiological and hydration indices were similar, pretrial heart rate, mean body temperature, serum osmolality, and body mass were also analysed in a similar manner. The effect of environment, work intensity, and their interaction were tested. Paired t-tests, using a Bonferroni correction, were conducted where significant differences were observed. All data was analysed using SPSS . Significance was set a priori at the P < 0.05 level.The data are displayed as mean ± SD unless otherwise stated. Assumption of normal distribution of data was assessed using descriptive methods  and inferential statistics (Shapiro-Wilk test). When the assumption of sphericity was violated, significance was adjusted using the Greenhouse-Geisser method to adjust the degrees of freedom to increase the critical values of the P = 0.213), mean body temperature (P = 0.176), serum osmolality (P = 0.407), or body mass (P = 0.894). The mean ± SD (range) duration of rest was 91 ± 18 min (58–155) when multiple trials were performed on the same day.Participants commenced all nine trials from a resting physiological baseline , with noAll twelve participants completed all nine trials  with no serious adverse events recorded. The majority of trials  were terminated due to participants' heart rate exceeding 90% of their maximum . A totalP < 0.001; 1 − β = 1.0), work intensity , and their interaction .A significant main effect in tolerance time , Table 2P < 0.001; 1 − β = 0.992), skin temperature , mean body temperature , heart rate , and body mass loss . Core temperature (P < 0.01), skin temperature (P < 0.05), and mean body temperature (P < 0.01) were lower at the end of the 5.5 km·h−1 trials compared to the 2.5 and 4 km·h−1 trials. Body mass loss was also greater in the lower work intensities . Conversely, despite a trend for an increase in heart rate at the 2.5 km·h−1 trials compared to the 4 (P = 0.055) and 5.5 (P = 0.077) km·h−1 trials, no post hoc differences were observed.Work intensity  had a siP < 0.001; 1 − β = 1.0), heart rate , and body mass loss differed across the three environments. Skin temperature (P < 0.001) and body mass loss (P = 0.027) were significantly higher in the WBGT21 condition compared to the WBGT37 environment. Skin temperature was also higher (P = 0.019) in the WBGT21 condition compared to the WBGT30. Heart rate was higher (P = 0.02) in the WBGT21 environment compared to the WBGT37 environment. The ambient environment had no significant effect on core temperature , mean body temperature , or PSI . No other statistically significance differences were observed.Skin temperature  physiological work tolerance is significantly influenced by the external environment and workload, (2) despite the short durations of exercise (~24 min), on average moderate to very high levels of physiological strain were experienced by the participants, and (3) cardiovascular, rather than thermoregulatory, strain is the limiting factor to work tolerance when wearing this ensemble.−2 or 500 W, as evident in the 5.5 km·h−1 trials, the role the environment plays in the rate of heat storage and work tolerance is limited [−2) across the three environments , and termination criteria [−1·min−1) were similar.The current study is in agreement with previous research findings examining PPE of similar weight , 19 and criteria . MoreoveConsidering the combined weight of the chemical protective clothing and respirator is minimal (~2 kg); it is unlikely that the additional weight alone was responsible for this reduction in work capacity. For example, Teitlebaum and Goldman  have preV˙O2max . MoreoveV˙O2max , 37. ConV˙O2max . This hyV˙O2max .It has been postulated that the impact of multiple clothing layers, particularly if associated with extra load carriage, may have greater effects on work tolerance than the added resistance of breathing through a respirator , 21. RegThe findings of this study are limited to young healthy males. Due to physiological differences between males and females  and younIn conclusion, this investigation has demonstrated that physiological tolerance times are significantly reduced in higher ambient environments and workloads when wearing explosive and chemical PPE. Secondly, despite the short durations of exercise, high to very high levels of physiological strain were experienced by all participants. Finally, cardiovascular strain is the limiting factor to work tolerance when wearing this heavy, multilayered, and encapsulating PPE.


Free nitrous acid (FNA), which is the protonated form of nitrite and inevitably produced during biological nitrogen removal, has been demonstrated to strongly inhibit the activity of polyphosphate accumulating organisms (PAOs). Herein we reported an efficient process for wastewater treatment, i.e., the oxic/anoxic/oxic/extended-idle process to mitigate the generation of FNA and its inhibition on PAOs. The results showed that this new process enriched more PAOs which thereby achieved higher phosphorus removal efficiency than the conventional four-step  biological nutrient removal process . It was found that this new process increased pH value but decreased nitrite accumulation, resulting in the decreased FNA generation. Further experiments showed that the new process could alleviate the inhibition of FNA on the metabolisms of PAOs even under the same FNA concentration. Enhanced biological phosphorus removal is one important strategy to protect natural waters from eutrophication. It is usually achieved through culturing an activated sludge with alternating anaerobic and oxic conditions, by which polyphosphate accumulating organisms (PAOs), the microorganisms responsible for phosphorus removal in wastewater treatment plants (WWTPs), can be largely enriched. To gain deep understandings regarding this biological phosphorus removal regime, numerous studies have been made in the past two decades123In general, biological phosphorus removal can be excellently achieved in well-defined laboratory experiments, with the high abundance of PAOs above 90%567−3 mg HNO2-N/L−3 mg HNO2-N/L could result in the complete loss of aerobic phosphorus uptake−3 mg HNO2-N/L. Anoxic phosphorus uptake was also significantly affected by FNA presence2-N/L would cause the complete loss of anoxic phosphorus uptake−3 mg HNO2-N/L. Due to the severe inhibition on PAOs caused by FNA and the massive quantity of wastewaters treated daily, any improvement for reducing FNA generation or mitigating its inhibition on PAOs in current methods should have tangible economic and ecological consequences.Nitrite is inevitably produced in substantial amounts during biological nitrogen removal. It was reported that nitrite concentration could accumulate up to 12.3–22.6 mg/L in domestic wastewater treatment processes511Several strategies, such as activated sludge adaption, pH adjustment, temperature control, and the feed flow and mode optimization, have been recommended to minimize the inhibitory effect of FNA on PAOs6Besides the widely accepted anaerobic/oxic (A/O) phosphorus removal regime, PAOs are verified to be also enriched readily in the oxic/extended-idle (O/EI) wastewater treatment regime161718The purpose of this paper is to report this efficient method for significantly mitigating the generation of FNA and its inhibition on PAOs. Firstly, a new BNR process is designed based on the recently exploited O/EI regime, and its feasibility of BNR is evaluated. Since the O/EI regime is a phosphorus removal process with low nitrogen removal performances (around 60%), an anoxic phase is inserted into the oxic phase to enhance nitrogen removal. Therefore, the new BNR process developed here is performed as the oxic/anoxic/oxic/extended-idle (O/A/O/EI) regime. Then, the performances of BNR and the abundances of PAOs between the new process and conventional four-step  BNR process are compared. Finally, the reasons for the new process showing higher abundance of PAOs are explored via the analysis of cyclic pH variation, nitrite accumulation, and changes of metabolic intermediates in PAO metabolisms.4+-N, NO2--N, NO3--N, and soluble orthophosphate (SOP) concentrations in the O/A/O/EI reactor during the long-term operation are illustrated in 4+-N, NO2--N, NO3--N, and SOP concentrations during stable operation were respectively maintained among 2.67–3.49, 0.15–0.31, 0.83–1.25 and 0.30–0.52 mg/L, which indicated that the efficiencies of nitrogen and phosphorus removal in the O/A/O/EI reactor were above 91% and 96%, respectively. The long-term experimental data showed that BNR could be successfully achieved in the new O/A/O/EI process.The data of the effluent NH4+-N, NO2--N, and NO3--N in the O/A/O/EI reactor were slightly lower than those in the A/O/A/O reactor, the efficiency of nitrogen removal between the two reactors was very close. However, the effluent SOP concentration in the O/A/O/EI reactor were much lower than that in the A/O/A/O reactor (0.41 ± 0.11 mg/L versus 2.70 ± 0.18 mg/L), which thereby caused a much higher phosphorus removal efficiency (97 ± 0.73% versus 82 ± 1.2%). FISH quantification further showed that the abundances of PAOs and glycogen accumulating organisms (GAOs) were respectively accounted for 41 ± 7% and 11 ± 3% in the O/A/O/EI reactor while the corresponding data in the A/O/A/O reactor were 30 ± 5% and 24 ± 4%, respectively  and pH might be the effect parameters since they are not constantly controlled during the whole process. Therefore, the cyclic variations of DO and pH between the two reactors during the steady-state operation were first compared, and the data are shown in 2 expelled from the reactor by air-stripping, while the following pH decline was probably ascribed to nitrification. It was reported that denitrification and phosphorus uptake were the primarily reasons for pH increase in the anoxic phase and oxic phase19The profile of pH change in the two reactors, however, exhibited obvious differences. In the O/A/O/EI reactor, pH gradually increased from 8.0 to 8.6 during the initial 60 min of first oxic phase and then decreased slightly during the remaining of this phase. In the following anoxic and oxic phases, a gradual increase to a final pH of 8.6 was observed. During the subsequent idle phase pH decreased gradually to the final pH of 8.2. In the A/O/A/O reactor, pH decreased from 8.0 to 7.4 in the anaerobic phase, and then a gradual increase followed by a slight decrease of pH was measured in the first oxic phase. In the subsequent anoxic phase, pH showed a gradual increase tendency then pH decreased slightly in the following oxic and idle phases. It can be clearly seen that cyclic variation of pH value in the O/A/O/EI reactor (8.0–8.6) was higher than that in the A/O/A/O reactor (7.2–8.2). The initial higher pH value achieved in the O/A/O/EI reactor might be mainly due to CO4+-N concentration was quickly decreased while NO2--N and NO3--N were substantially accumulated, with NO2--N and NO3--N accumulation up to 6.4 and 6.1 mg/L, respectively. In the subsequent anoxic phase, SOP and NH4+-N concentrations decreased slightly whereas NO2--N and NO3--N were largely decreased, suggesting that denitrification occurred in this period. Then, after 30 min of oxic phase , SOP, NH4+-N, NO2--N, and NO3--N concentrations in the effluent were 0.40, 3.0, 0.23, 0.97 mg/L, respectively. As comparison, it can be observed that a substantial amount of SOP was released in the anaerobic phase of conventional A/O/A/O reactor, then SOP uptake, NH4+-N oxidation, and NOx--N accumulation took place concurrently. During the subsequent anoxic phase, nitrate and nitrite reductions were clearly measured. After 30 min of oxic phase , SOP, NH4+-N, NO2--N, and NO3--N concentration in the effluent were 2.7, 3.1, 0.29, 1.12 mg/L, respectively. Those behaviors were similar to the observations in the previous publications25More importantly, the concentration of severe inhibitor to PAOs, i.e., FNA, is reported to closely relevant to pH value−4 mg HNO2-N/L whereas the corresponding datum was 0.24 × 10−3 mg HNO2-N/L in the A/O/A/O reactor. The maximum FNA concentration in the A/O/A/O reactor was approximately 4.6-time higher than that in the O/A/O/EI reactor. Similar observations were also observed in other cycles. It was reported that aerobic SOP uptake was severely affected when FNA was 0.26 × 10−3 mg HNO2-N/L2 prior to nitrite was the main electron acceptor in this study, which might be the reason for the inconsistent results.It should be highlighted that the maximal nitrite accumulation in the O/A/O/EI reactor was lower than that in the conventional A/O/A/O reactor (6.4 versus 7.5 mg/L), though the two reactors had approximately same effluent nitrite concentration. In addition, it can be found that pH value at the time for the O/A/O/EI reactor achieving its maximal nitrite accumulation was higher than that for the A/O/A/O reactor (8.4–8.5 versus 7.9). According to the formula proposed by Anthonisen et al.2 for PHAs formation were respectively provided via poly-P hydrolysis and glycogen degradation, the TCA cycle seems to supply both ATP and NADH2 for PHAs synthesis in the O/A/O/EI reactor since it is generally accepted that the TCA cycle will dominate under aerobic conditions. However, it is still unclear why the O/A/O/EI reactor can alleviate the inhibition of FNA on the metabolisms of PAOs, as the TCA cycle plays an important role in PAO metabolisms of both regimes. Further efforts need to be carried out in future.FNA can inhibit or inactivate the activities of some key enzymes relevant to phosphorus removal. For instance, glyceraldehyde-3-phosphate dehydrogenase (GADP) and sulfhydryl (SH)-containing enzymes, which are respectively key enzymes involved in both glycolysis (gluconeogenesis) and the tricarboxylic acid (TCA) cycle, are reported to be heavily inhibited through reaction with FNA . The traThis paper presents an effective strategy for mitigating the generation of FNA and its inhibition on PAOs. That is, by modifying the wastewater treatment operation regime as the O/A/O/EI regime the abundance of PAOs and the efficiency of phosphorus removal can be significantly improved. This was experimentally demonstrated via a long-term test in two reactors operated as the new O/A/O/EI regime and the conventional A/O/A/O regime, respectively. The abundance of PAOs cultured in the O/A/O/EI reactor was about 11% higher than those in the conventional A/O/A/O reactor, which led to 15% of improved phosphorus removal efficiency. Moreover, this wastewater treatment regime based strategy did not decrease but slightly increase the nitrogen removal performance. Considering the huge quantities of wastewater treated daily, this strategy has a significant consequence from an ecological perspective.Compared with other strategies such as pH adjustment and temperature control6It should be noted that although the hydraulic retention time (HRT) between the O/A/O/EI and A/O/A/O reactors operated in this studies was maintained the same, the HRT controlled in the O/A/O/EI regime may slightly higher than that in the conventional BNR regime, because one cycle of the conventional BNR systems can be shortened to be 6 h via process optimization whereas the O/A/O/EI regime needs a relatively long idle period to enrich PAOs . This characteristic implies that the proposed O/A/O/EI regime will increase the volumes of bioreactors when treating the same amount of wastewater. However, this drawback can be settled via reactor reconfiguration as proposed in 2PO4 was selected as the phosphorus source. The chemical oxygen demand (COD) and orthophosphate (PO43--P) concentrations in the wastewater were approximately maintained at 300 and 15 mg/L, respectively. Hence, the ratio of COD: PO43--P in the influent was controlled at 20 mg COD/(mg PO43--P), which was considered as being favorable to the growth of PAOs4Cl, 0.5 mg CaCl2, 0.5 mg MgSO4, and 1 mL of a trace metals solution. The trace metals solution had been described in our previous publicationSynthetic wastewater used throughout these investigations, unless otherwise described, was the same and prepared daily. Acetate was used for the sole carbon source since it was the most common volatile fatty acids present in real domestic wastewaters2/O process. The initial concentration of mixed liquor suspended solids (MLSS) was 3800 mg/L and mixed liquor volatile suspended solid (MLVSS) was 2400 mg/L. The activated sludge was maintained at 20 ± 0.5°C in a temperature controlled room. One reactor was performed as the developed O/A/O/EI regime while the other was operated as the classical four-step A/O/A/O regime in parallel. Both reactors were operated with three 8-h cycles daily, and each 8-h cycle of the O/A/O/EI regime consisted of a 120 min oxic phase, a 90 min anoxic phase, a 30 min oxic phase, a 30 min settling and decanting phase, and a 210 min idle phase. As comparison, the conventional four-step BNR regime was also operated according to the literature with minor revision31This study was conducted in two identical sequencing batch reactors with a working volume of 12 L each. Both reactors were seeded with activated sludge obtained from a WWTP in Changsha, PR China, which was operated as A4+-N, and NOx--N.It should be noted that during the idle phase mixture stirring was not conducted in the routine operation of both reactors, but when cyclic tests were carried out, both reactors were mixed with a mechanical stirrer (150 rpm) to facilitate sampling. The mixed liquor samples were taken every 30 min and immediately filtered through a Whatmann GF/C glass microfiber filter (1.2 μm). The sludge sample was used to assay for MLSS, MLVSS, PHAs, and glycogen. The filtrate was used for the analyses of SOP, COD, NH2+, Mg2+ are present in wastewater. With the increase of pH the chemical phosphorus precipitation was enhancedPhosphorus can be removed via chemical precipitation when some metal ions such as Ca4+-N, NOx--N, SOP, and COD. Then, they were resuspended in tap water with a final volume of 1.2 L each before being evenly divided into six reactors. The two groups of reactors (six each) were respectively operated as the same as the A/OA/O and O/A/O/EI reactors expect for the following differences. Allyl-Nthiourea (a nitrification inhibitor) was added at a concentration of 2 mg/L to each reactor to inhibit the nitrification according to the literature−3, 0.15 × 10−3, 0.26 × 10−3, 0.38 × 10−3, and 0.51 × 10−3 mg HNO2-N/L through controlling nitrite concentration, pH, and temperature. It was reported that the FNA concentration could be calculated by the formula SN-NO2/(Ka × 10pH) with Ka value determined by the formula Ka = e(−2300/(T + 273)) for a given temperature T (°C)The A/OA/O reactor is developed from the conventional A/O phosphorus removal regime whereas the O/A/O/EI reactor is developed from the recently exploited O/EI regime, thus it is necessary to investigate whether there are different effects on PAO metabolisms between the two reactors even under the same FNA levels. The following batch experiment was executed to provide such support. Two identical sludge mixtures (2.4 L each) were respectively withdrawn from a WWTP in Changsha, PR China. The mixtures were centrifuged (5000 rpm for 5 min) and washed three times with tap water to remove the residual NHCOD, SOP, nitrite, nitrate, ammonia, MLSS, and MLVSS were measured by standard methodsThe fluorescence in situ hybridization (FISH) with 16s rRNA-targeted oligonucleotide probes was carried out to quantify the abundances of PAOs and GAOs, and the methods were the same as described in the literatureJ.W.Z. carried out the experiments and drafted the paper, D.B.W. and X.M.L. designed the experimental plan and revised the paper, Q.Y., H.B.C., Y.Z., H.X.A. and G.M.Z. analyzed the data. All authors contributed to the scientific discussion.Supporting Information