Expanding Color Control of Anodically Coloring Electrochromes Based on Electron-Rich 1,4-Dihydropyrrolo[3,2-b]pyrroles.

Anodically coloring electrochromes have received attention in recent years as high-contrast alternatives to cathodically coloring electrochromes due to their superior optical contrast during electrochemical switching. While current systems represent significant progress for organic electrochromics, it is necessary to expand the structural diversity of these materials while simultaneously reducing the hazards associated with synthetic protocols. With these considerations in mind, a family of 1,4-dihydropyrrolo[3,2-b]pyrrole (DHPP) chromophores with varying functionalities along the 2,5-axis was envisioned to accomplish these goals. After predicting different absorbance traits as oxidized molecules with time-dependent density functional theory, DHPP chromophores with varying peripheral functionalities were synthesized in a single aerobic synthetic step via an iron-catalyzed multicomponent reaction and characterized as high-contrast chromophores. In solution, the DHPP chromophores absorb in the ultraviolet region of the electromagnetic spectrum, resulting in color-neutral L*a*b* color coordinates of ∼100, 0, 0. Upon chemical oxidation, each molecule transitions to absorb at various points across the visible spectrum based on the extent of electron-donating ability and can display five distinct colors. Importantly, the chromophores are redox-active and display switching capabilities with an applied electrochemical potential. In conjunction with building fundamental insights into molecular design of DHPP chromophores, the results and synthetic simplicity of DHPPs make them compelling materials for color-controlled high-contrast electrochromes.

Baseline physiological data from anesthetized pigs in a VX intoxication model.

Over the past fifty years, swine models have been used for organophosphorus intoxication studies. Among these studies and others on the swine model in general, some physiological data, especially cholinesterase activity highly impacted by organophosphorus compounds like nerve agent VX, still need to be completed. To support and compare our model to others, we have published the experimental protocol, the physiological values of 31 juvenile anesthetized pigs, and the 6 h-follow-up of six supplementary anesthetized control animals and 7 VX-intoxicated pigs. We reported hemodynamics and respiratory parameters, blood levels in several biochemical parameters, blood gas, and complete blood count and compared them to the literature. We also focused on tissue and blood cholinesterase activities and detailed them for acetylcholinesterase and butyrylcholinesterase. After establishing a broad physiological data set consistent with the literature, we reported several cardio-respiratory parameters that seem more affected by an organophosphate intoxication, like heart rate, arterial blood pressure, cardiac output, and respiratory rate. Within the blood, oxygen saturation (SpO2), lactatemia, base excess, and glycemia can also be measured and associated with the other parameters to evaluate the life-threatening status. This swine model is currently used to develop and evaluate medical countermeasures against organophosphate nerve agent intoxications.

Development of simple, scalable protease production from Botrytis cinerea.

Heat haze-forming proteins are stable during winemaking and are typically removed via adsorption to bentonite. Proteolytic degradation is an alternative method to prevent wine-haze and offers the opportunity to reduce the environmental impacts and labor cost of the process. Herein, we describe the development of a production system for Botrytis cinerea proteases for the enzymatic degradation of heat haze-forming proteins. The effect of culture medium on the secretion of glucan by B. cinerea was investigated and methods to inactivate B. cinerea laccase in liquid culture medium were assessed. Protease production by B. cinerea was scaled up from 50 mL in shake flasks to 1 L in bioreactors, resulting in an increase in protease yield from 0.30 to 3.04 g L-1. Glucan secretion by B. cinerea was minimal in culture medium containing lactose as a carbon source and either lactic or sulfuric acid for pH control. B. cinerea laccases were inactivated by reducing the pH of culture supernatant to 1.5 for 1 h. B. cinerea proteases were concentrated and partially purified using ammonium sulfate precipitation. SWATH-MS identified aspartic acid protease BcAP8 amongst the precipitated proteins. These results demonstrate a simple, affordable, and scalable process to produce proteases from B. cinerea as a replacement for bentonite in winemaking. KEY POINTS: • Isolates of B. cinerea that produce proteases with potential for reducing wine heat-haze forming proteins were identified. • Media and fermentation optimization increased protease yield tenfold and reduced glucan secretion. • Low pH treatment inactivated laccases but not proteases.

Effective degradation of polychlorinated biphenyls by a facultative anaerobic bacterial consortium using alternating anaerobic aerobic treatments.

Polychlorinated biphenyls (PCBs) are synthetic mixtures of chlorinated hydrocarbon compounds that were widely used in the past and still found in the environment due to their highly recalcitrant nature. A combination of anaerobic dechlorination and aerobic oxidation is essential to degrade these PCB mixtures into less toxic products. It was hypothesized that due to the complexity of PCB mixtures, a consortium of carefully selected suitable microbial species will perform better than the application of individual microbes. In the present study, biodegradation of the commercial PCB mixture, Aroclor 1260, was studied under two different combined anaerobic-aerobic conditions, namely, alternating (AN) and two stage (TS). The facultative anaerobic bacterial consortium consisted of naturally occurring Achromobacter sp. NP03, Ochrobactrum sp. NP04 and Lysinibacillus sp. NP05. These bacteria were found capable as individuals of solubilizing and degrading PCBs under both anaerobic and aerobic conditions. 49.2 ±â€¯2.5% total reduction of the original 50 mg/L Aroclor 1260 mixture was achieved after 2 weeks in AN treatment whereas the reduction was only 24.44 ±â€¯2.46% in TS treatment. At the end of week 6, a yield of 17.63 ±â€¯0.91 mg/L chloride released was measured under AN condition compared to 11.79 ±â€¯1.28 mg/L measured under TS condition. The overall results suggested that the microbial consortia capable of degrading and utilizing PCBs under both, anaerobic and aerobic conditions achieved better PCB degradation by repeated exposure to short periods of anaerobic and aerobic conditions alternatingly rather than the conventional two stage anaerobic-aerobic conditions.

Co-utilization of acidified glycerol pretreated-sugarcane bagasse for microbial oil production by a novel Rhodosporidium strain.

Acidified glycerol pretreatment is very effective to deconstruct lignocellulosics for producing glucose. Co-utilization of pretreated biomass and residual glycerol to bioproducts could reduce the costs associated with biomass wash and solvent recovery. In this study, a novel strain Rhodosporidium toruloides RP 15, isolated from sugarcane bagasse, was selected and tested for coconversion of pretreated biomass and residual glycerol to microbial oils. In the screening trails, Rh. toruloides RP 15 demonstrated the highest oil production capacity on glucose, xylose, and glycerol among the 10 strains. At the optimal C:N molar ratio of 140:1, this strain accumulated 56.7, 38.3, and 54.7% microbial oils based on dry cell biomass with 30 g/L glucose, xylose, and glycerol, respectively. Furthermore, sugarcane bagasse medium containing 32.6 g/L glucose from glycerol-pretreated bagasse and 23.4 g/L glycerol from pretreatment hydrolysate were used to produce microbial oils by Rh. toruloides RP 15. Under the preliminary conditions without pH control, this strain produced 7.7 g/L oil with an oil content of 59.8%, which was comparable or better than those achieved with a synthetic medium. In addition, this strain also produced 3.5 mg/L carotenoid as a by-product. It is expected that microbial oil production can be significantly improved through process optimization.

Solubilization and degradation of polychlorinated biphenyls (PCBs) by naturally occurring facultative anaerobic bacteria.

A combination of solubilization and degradation is essential for the bioremediation of environments contaminated with complex polychlorinated biphenyls (PCB) mixtures. However, the application of facultative anaerobic microorganisms that can both solubilize and breakdown hydrophobic PCBs in aqueous media under both anaerobic and aerobic conditions, has not been reported widely. In this comprehensive study, four bacteria discovered from soil and sediments and identified as Achromobacter sp. NP03, Ochrobactrum sp. NP04, Lysinibacillus sp. NP05 and Pseudomonas sp. NP06, were investigated for their PCB degradation efficiencies. Aroclor 1260 (50 mg/L), a commercial and highly chlorinated PCB mixture was exposed to the different bacterial strains under aerobic, anaerobic and two stage anaerobic-aerobic conditions. The results confirmed that all four facultative anaerobic microorganisms were capable of degrading PCBs under both anaerobic and aerobic conditions. The highest chlorine removal (9.16 ±â€¯0.8 mg/L), PCB solubility (14.7 ±â€¯0.93 mg/L) and growth rates as OD600 (2.63 ±â€¯0.22) were obtained for Lysinibacillus sp. NP05 under two stage anaerobic-aerobic conditions. The presence of biosurfactants in the culture medium suggested their role in solubility of PCBs. Overall, the positive results obtained suggest that high PCB hydrolysis can be achieved using suitable facultative anaerobic microorganisms under two stage anaerobic-aerobic conditions. Such facultative microbial strains capable of solubilization as well as degradation of PCBs under both anaerobic and aerobic conditions provide an efficient and effective alternative to commonly used bioaugmentation methods utilizing specific obligate aerobic and anaerobic microorganisms, separately.

Placental structure and function in different breeds in horses.

Ponies and sometimes draft horses are often used as experimental models for horses although size and metabolic parameters are known to vary between horse breeds. So far, there is little information about differences of placental structure and no information about differences of placental function between breeds. The aim of this study was to investigate differences in placental size, structure and function at birth in relation to foal size and weight in ponies, Saddlebred and draft horses. Pony, Saddlebred and draft horse pregnancies were obtained by artificial insemination over 2 successive breeding seasons. Foals and total fetal membranes (TFM) were weighed and placentas measured for surface area at term. Placentas were sampled above the umbilical cord insertion. Surface density and volume fraction of the different cellular components of the placenta were measured on histological sections using stereology. The expression of genes involved in growth and development, nutrient transfer and vascularization was compared between groups. Foals and TFM were lighter at birth in ponies than Saddlebred horses, and both were lighter compared to draft horses. The surface density and volume fraction of microcotyledonary vessels was increased in pony compared to Saddlebred placentas. The relative expression of genes involved in growth and development was different between breeds and increased with maternal, fetal and placental weight. Primiparous dams produced lighter foals and smaller placentas, associated with a decreased volume fraction of microcotyledonary vessels and genes involved in growth and development and vascularization. Foal sex had little effect on placental structure and function as the expression of only one gene differed according to sex, with EGFR expression being decreased in placentas of females compared to males. In conclusion, foal and placental weight, as well as placental expression of genes involved in growth and development were correlated with maternal size. Placental structure also differed between breeds, with a stronger difference between ponies and both breeds of horses.

Placental alterations in structure and function in intra-uterine growth-retarded horses.

BACKGROUND: Following embryo transfer (ET), the size and breed of the recipient mare can affect fetal development and subsequent post natal growth rate and insulin sensitivity in foals. OBJECTIVES: To investigate placental adaptation in pregnancies where increased or restricted fetal growth was induced through ET between Pony, Saddlebred and Draught horses. STUDY DESIGN: In vivo experiment. METHODS: Control Pony (P, n = 21) and Saddlebred (S, n = 28) pregnancies were obtained by artificial insemination. Increased pregnancies were obtained by transferring Pony (P-D, n = 6) and Saddlebred (S-D, n = 8) embryos into Draught mares. Restricted pregnancies were obtained by transferring Saddlebred embryos into Pony mares (S-P, n = 6). Placental weight and surface were recorded and samples collected for stereology and analysis of expression of genes involved in placental growth, vascularisation and nutrient transport. Data were analysed by linear model. RESULTS: S-P foals were growth retarded when compared with controls despite increased gestational length. Placental weight was reduced but placental surface density and volume fraction were increased. Placental expression of genes involved in growth and development and nutrient transfer was strongly reduced. In contrast, placental size and weight were increased in enhanced growth P-D and S-D foals. The trophoblastic surface density and the allantoic vessels surface density were decreased in P-D and S-D, respectively, both with very few modifications in gene expression. MAIN LIMITATIONS: Control embryos were produced by artificial insemination whereas experimental embryos were produced by ET. CONCLUSIONS: Placental structure and gene expression are modified after ET into a smaller or larger breed than that of the embryo. These adaptations contribute to the observed phenotype of foal growth restriction or enhanced growth at birth.

A proteomic view into infection of greyback canegrubs (Dermolepida albohirtum) by Metarhizium anisopliae.

Metarhizium anisopliae is a naturally occurring cosmopolitan fungus infecting greyback canegrubs (Dermolepida albohirtum). The main molecular factors involved in the complex interactions occurring between the greyback canegrubs and M. anisopliae (FI-1045) were investigated by comparing the proteomes of healthy canegrubs, canegrubs infected with Metarhizium and fungus only. Differentially expressed proteins from the infected canegrubs were subjected to mass spectrometry to search for pathogenicity related proteins. Immune-related proteins of canegrubs identified in this study include cytoskeletal proteins (actin), cell communication proteins, proteases and peptidases. Fungal proteins identified include metalloproteins, acyl-CoA, cyclin proteins and chorismate mutase. Comparative proteome analysis provided a view into the cellular reactions triggered in the canegrub in response to the fungal infection at the onset of biological control.

Identification of two novel xylanase-encoding genes (xyn5 and xyn6) from Acrophialophora nainiana and heterologous expression of xyn6 in Trichoderma reesei.

Two novel genes, xyn5 and xyn6, coding for family 11 xylanases, were isolated from the thermotolerant filamentous fungus, Acrophialophora nainiana, by PCR using degenerate primers. The xyn6 gene was further expressed in Trichoderma reesei. DNA sequence analysis of xyn6 revealed an open reading frame (ORF) of 708 bp, interrupted by an intron of 58 bp. The xyn6 ORF encodes a predicted protein of 236 amino acid residues. The mature recombinant XynVI protein had a molecular mass of about 19 kDa, as estimated by gel electrophoresis. Analysis of the predicted amino acid sequence of XynVI paves the way for rational protein engineering by site-directed mutagenesis aiming to increase the thermostability of the heterologously-expressed xylanase.

Improvement of the secretion of extracellular proteins and isolation and characterization of the amylase I (amy1) gene from Ophiostoma floccosum.

UV mutagenesis was applied to improve protein secretion in Ophiostoma floccosum. Amylase activity was used as an indicator for enhanced protein production after repeated rounds of mutagenic treatment. The amylase activity in the culture supernatant of the best mutant (MQ.5.1) was increased by more than 240-fold compared to the initial parental strain. At the same time, the increase in total secreted protein was about six times greater than the parental strain. Secreted proteinase and lipase activities of the parental strain and four key mutants were also investigated. N-terminal sequencing of the five dominant protein bands separated by SDS-PAGE from the culture supernatant was conducted. Two of the proteins identified were subtilisin-like proteinases and one was a pepsin-like proteinase. In addition, one protein was identified as an alpha-amylase and one remained unidentified. A 6.5 kb DNA fragment was isolated by Genomic Walking PCR using primers based on the alpha-amylase amino acid sequence. The amplified fragment contained the entire gene encoding alpha-amylase (amy1) and its regulatory sequences. Analysis showed that multiple transcripts were generated from the single alpha-amylase gene locus.

Heterologous protein expression in filamentous fungi.

Filamentous fungi are commonly used in the fermentation industry for the large-scale production of proteins--mainly industrial enzymes. Recent advances in fungal genomics and related experimental technologies such as gene arrays and proteomics are rapidly changing the approaches to the development and use of filamentous fungi as hosts for the production of both homologous and heterologous gene products. The emphasis is moving towards sourcing new genes of interest through database mining and unravelling the circuits related to fungal gene regulation, applying, for example, transcriptomics. However, although heterologous fungal proteins are efficiently expressed, expression of gene products from other organisms is subject to several bottlenecks that reduce yield. Current approaches emphasize the study of pathways involved in protein modification and degradation in general rather than gene-by-gene approaches.

Isolation, characterization and expression of the hex1 gene from Trichoderma reesei.

Polymers of the HEX1 protein produce Woronin bodies in filamentous fungi. We have isolated and sequenced the hex1 gene and flanking regions from the industrially exploited fungus Trichoderma reesei. Multiple transcription start sites (TSS) and the 5' untranslated region (UTR) were identified by 5'RACE PCR. There are three hex1 transcript types, two of which originate from two TSSs at approximately -320 and -1335 from the start codon, which are separated by a 500-bp intron within the 5'UTR. The third transcript type results from alternative splicing of the intron within the coding sequence at the 3' end, which results in the inclusion or exclusion of an unconserved histidine-rich coding region. The three transcripts code for two forms of HEX1 protein. N-terminal sequencing of HEX1 separated by 2D gel electrophoresis confirms that there are two forms of HEX1 protein which are modified further by alternative cleavage of the N-terminus. The dominant form of HEX1 is coded by a cDNA with TSS at position -1335. Expression of hex1 on cellulase-inducing medium peaks strongly within 24 h of growth but the protein is expressed at a lower and more consistent level in medium containing glucose. This is the first investigation of expression of the hex1 gene encoding a protein unique to filamentous fungi.

Early nocturnal blood pressure changes in diabetic autonomic neuropathy assessed by Fourier series.

The 24 h periodic pattern of blood pressure was studied in 44 patients with diabetes mellitus (14 type 1, 30 type 2; mean duration of disease 6.5 +/- 1.8 years) in good metabolic control but with abnormal cardiovascular reflex responses; of these 21 were normotensive and 23 hypertensive. All had abnormal responses to at least two out of four tests: deep breathing, lying to standing, Valsalva manoeuvre and postural hypotension. Two sex- and age-matched groups, consisting of 20 normotensive and 20 hypertensive diabetic patients without dysautonomia, were studied as controls. Each patient underwent ambulatory blood pressure monitoring for at least 24 h, using an auscultatory automatic device. Data were analysed using the sum of three periodic functions (Fourier partial sum). In the diabetic normotensive groups, the absolute blood pressure fell to its night-time minimum more rapidly, and increased to its morning maximum more slowly, in those with abnormal cardiovascular reflexes than in the controls (nightly blood pressure decrease -5.8/-4.7 vs. -3.8/-4.0 mmHg/h; increase 4.7/3.6 vs. 5.9/6.1 mmHg/h). The same behaviour was found in both hypertensive groups but the amplitude of the differences was more marked (blood pressure nocturnal decrease -7.7/-7.1 vs. -4.3/-3.9 mmHg/h; increase 3.2/2.1 vs. 5.8/4.3 mmHg/h). This analysis of 24 h ambulatory blood pressure data may be of value in diagnosis and evaluation of autonomic deficits.

[A validation of the data obtained with the simultaneous recording of blood pressure and the 24-hour electrocardiogram]. / Validazione dei dati ottenuti con registrazione contemporanea della pressione arteriosa e dell'elettrocardiogramma per 24 ore.

Aim of this study was to evaluate the blood pressure (BP) measurement reliability of a light weight ambulatory BP and ECG recorder. Micro AM is a new 300 g portable apparatus that combines in one device both the ambulatory BP and solid state ECG recording. The dimensions of the Micro AM are 75 x 140 x 29 mm. The monitor measures BP using Korotkoff phase 1 for systolic and phase 5 for diastolic BP, and concurrently measures oscillometric BP, one method validating the other. In addition, the manual and programmed BP measurement modes can be supplemented by an "intelligent" mode in which the ECG triggers an ambulatory BP reading during an abnormal ST segment change. A standard mercury manometer was connected with the cuff of the Micro AM with a Y-shaped part, and 12 BP measurements were simultaneously taken at 5 min intervals by the automatic device in auscultatory mode and by a trained technician in 86 normotensive volunteers (aged from 18 to 44 years, 37 males and 49 females). The algebraic differences, the frequency distribution and the difference distribution of systolic and diastolic data between the 2 methods were calculated. The results show that the automatic method gives values for systolic BP that are lower than conventional ones (average differences -0.643 mmHg), whereas for diastolic BP, the values are higher (average differences +0.229 mmHg). Then, Student's paired t-test was used to evaluate statistically significant differences. The test relative to systolic BP was significant to the critical level of 0.1%, but the differences being 3 times smaller than the instrumental tolerance. On the contrary, diastolic BP differences were non significant. In conclusion, we found a good agreement between BP recorded automatically and by sphygmomanometer.

Plasma atrial natriuretic peptide in young normotensive subjects with a family history of hypertension and in young hypertensive patients.

Plasma atrial natriuretic peptide (ANP) behavior was evaluated in 26 untreated essential hypertensives, 21 normotensives, and 20 normotensives with hypertensive heredity under normal sodium intake (120 mEq of Na+/day). All subjects were men, mean age 22.1 +/- 1.9 years. Plasma ANP was evaluated by radioimmunoassay on samples collected in supine position upon waking and again after 1 h of orthostatism. Resulting data showed that ANP in hypertensives (supine = 44.5 +/- 19.4 pg/mL, orthostatism = 24.1 +/- 11.6 pg/mL) was at higher levels than in controls (supine = 38.3 +/- 19.4 pg/mL, orthostatism = 19.9 +/- 10.6 pg/mL) or in normotensives with hypertensive heredity (supine = 42.1 +/- 16.8 pg/mL, orthostatism = 23.2 +/- 10.8 pg/mL). Mean ANP level was higher in the latter group than in the control group (supine = +9%; orthostatism = +14.2%). In conclusion, plasma ANP is raised in young essential hypertensives, resulting in slightly elevated levels in normotensives with hypertensive heredity.

[Relation of atrial natriuretic peptide and endogenous digoxin-like activity in the plasma of young hypertensive patients]. / Rapporti tra peptide atriale natriuretico e attività digossinosimile endogena nel plasma di pazienti giovani ipertesi.

The relationships between plasma digoxin-like immunoreactivity (DLS) and atrial natriuretic peptide (ANP) were investigated in 10 young essential hypertensives (mean age 22 +/- 2 years) and in 10 normotensives. In young hypertensives, plasma DLS and ANP were at an average level of 31.2 +/- 8 pg/ml and 56.7 +/- 20 pg/ml respectively, showing a significant correlation (r = 0.66; p less than 0.05). In normal subjects plasma DLS and ANP were at an average level of 19.1 +/- 8 pg/ml and 37.6 +/- 16.7 pg/ml respectively (n.s.). Plasma DLS and ANP were higher in hypertensives than in normotensives (p less than 0.01). In conclusion, DLS and ANP appear to have a similar behaviour, possibly due to their modulation by common stimuli.

[Effects of active orthostatism on blood levels of atrial natriuretic peptide in the healthy subject]. / Effetti dell'ortostatismo attivo sui livelli plasmatici di peptide atriale natriuretico nel soggetto normale.

The effects of active changes in posture, from recumbency to upright position (60 minutes), on the circulating levels of atrial natriuretic peptide (ANP) in healthy human subjects were studied by using a radioimmunoassay method. In supine position, plasma ANP levels ranged from 12 pg/ml to 51.5 pg/ml, with an average level of 35.3 +/- 11.5 pg/ml. After 1 hour of orthostatic position, plasma ANP levels varied from 10 pg/ml to 35 pg/ml, with an average level of 21 +/- 11.5 pg/ml. These results suggest that ANP is involved in the hemodynamic modifications following postural stimuli. Thus, postural changes can be taken in to account for evaluating plasma ANP behaviour properly.