A Ser29Leu Substitution in the Cytosine Deaminase Fca1p Is Responsible for Clade-Specific Flucytosine Resistance in Candida dubliniensis

The population structure of the opportunistic yeast pathogen Candida dubliniensis is composed of three main multilocus sequence typing clades (clades C1 to C3), and clade C3 predominantly consists of isolates from the Middle East that exhibit high-level resistance (MIC₅₀ ≥ 128 μg/ml) to the fungicidal agent flucytosine (5FC). The close relative of C. dubliniensis, C. albicans, also exhibits clade-specific resistance to 5FC, and resistance is most commonly mediated by an Arg101Cys substitution in the FUR1 gene encoding uracil phosphoribosyltransferase. Broth microdilution assays with fluorouracil (5FU), the toxic deaminated form of 5FC, showed that both 5FC-resistant and 5FC-susceptible C. dubliniensis isolates exhibited similar 5FU MICs, suggesting that the C. dubliniensis cytosine deaminase (Fca1p) encoded by C. dubliniensis FCA1 (CdFCA1) may play a role in mediating C. dubliniensis clade-specific 5FC resistance. Amino acid sequence analysis of the CdFCA1 open reading frame (ORF) identified a homozygous Ser29Leu substitution in all 12 5FC-resistant isolates investigated which was not present in any of the 9 5FC-susceptible isolates examined. The tetracycline-inducible expression of the CdFCA1 ORF from a 5FC-susceptible C. dubliniensis isolate in two separate 5FC-resistant clade C3 isolates restored susceptibility to 5FC, demonstrating that the Ser29Leu substitution was responsible for the clade-specific 5FC resistance and that the 5FC resistance encoded by FCA1 genes with the Ser29Leu transition is recessive. Quantitative real-time PCR analysis showed no significant difference in CdFCA1 expression between 5FC-susceptible and 5FC-resistant isolates in either the presence or the absence of subinhibitory concentrations of 5FC, suggesting that the Ser29Leu substitution in the CdFCA1 ORF is the sole cause of 5FC resistance in clade C3 C. dubliniensis isolates.Candida dubliniensis is an opportunistic yeast pathogen that was first described in 1995 in human immunodeficiency virus-infected patients in Ireland (39). Since then the organism has been shown to have a worldwide distribution and has been recovered from other groups of immunocompromised individuals and from patients with severe underlying disease (2-4, 11, 29, 30, 36-38, 44). The population structure of C. dubliniensis has previously been investigated by using the species-specific complex DNA fingerprinting probe Cd25 and multilocus sequence typing (MLST) (4, 11, 15, 18). Early Cd25 fingerprinting analyses demonstrated that C. dubliniensis consists of two fingerprinting groups, termed Cd25 group I and Cd25 group II (15). Group I isolates comprise the majority of isolates investigated from many countries around the world and are very closely related, with an average similarity coefficient value (S_AB) of 0.8. Group II isolates are less closely related and have an average S_AB value of 0.47 (15). These results were later confirmed with a larger collection of isolates by Gee et al. (11), who also showed that Cd25 group I isolates comprised a single genotype (genotype 1) on the basis of sequence analysis of the internal transcribed spacer (ITS) region of the ribosomal DNA operon. Furthermore, Cd25 group II isolates were found to belong to three ITS genotypes (genotypes 2 to 4). In 2005, a study by Al Mosaid et al. (4) identified a third Cd25 fingerprinting group, termed Cd25 group III, which exhibited an average S_AB value of 0.35, among C. dubliniensis isolates recovered exclusively in Egypt, Saudi Arabia, and Israel, all of which belonged to ITS genotypes 3 or 4. All isolates belonging to Cd25 group III examined exhibited high-level intrinsic resistance to the antifungal drug flucytosine (5FC), apart from one Israeli isolate that was 5FC susceptible. This phenotype did not occur in isolates belonging to either Cd25 group I or Cd25 group II, including isolates from Cd25 groups I and II recovered from Egypt, Saudi Arabia, and Israel (4). Recent studies that have used MLST analysis to investigate the population structure of C. dubliniensis revealed the presence of three distinct clades, termed clades C1 to C3 (18). All 5FC-resistant isolates belonging to Cd25 fingerprint group III were found to cluster exclusively in MLST clade C3 (18). More recently, MLST was used to show that clade C1 C. dubliniensis isolates recovered from avian excrement-associated samples were genetically distinct from other clade C1 isolates that were recovered from humans (19).The closest relative of C. dubliniensis, Candida albicans, also exhibits clade-specific resistance to 5FC, with 72.7% of isolates in MLST clade C1 (Ca3 fingerprinting clade I) exhibiting reduced susceptibility to this antifungal agent (23, 32). In C. albicans, the 5FC resistance patterns vary among isolates and range from reduced susceptibility (MICs, 0.5 to 2 μg/ml) to intermediate resistance (MICs, 2 to 8 μg/ml) or high-level resistance (MICs, ≥8 μg/ml); and a wide range of 5FC MICs for this drug have been reported among isolates (range, 0.06 μg/ml to ≥128 μg/ml) (7, 13). In C. dubliniensis, the resistance patterns are more clearly defined, with 5FC-susceptible isolates exhibiting 5FC MICs of ≤0.125 μg/ml and 5FC-resistant isolates exhibiting 5FC MICs of ≥128 μg/ml (4). To date, 5FC resistance in C. dubliniensis has been reported only in isolates from the Middle East, all of which that have been tested belong to MLST clade C3 (1, 4, 18, 29).The antifungal action of 5FC relies on the intracellular conversion of 5FC to fluorouracil (5FU) by cytosine deaminases upon entry into fungal cells (Fig. ​(Fig.1).1). Cytosine deaminase (Fca1p) is encoded by FCA1 in C. albicans and C. dubliniensis (CdFCA1) (4, 9), and the FCA1 genes in these two species are homologues of the FCY1 gene in Saccharomyces cerevisiae (9) and in other Candida species, such as Candida lusitaniae (26). The absence of cytosine deaminases in mammalian cells prevents 5FC toxicity in humans, as the 5FC prodrug itself is nontoxic. After the conversion of 5FC to 5FU, the FUR1-encoded uracil phosphoribosyltransferase (UPRT) catalyzes the phosphorylation of 5FU to fluorouridine monophosphate (5FUMP) (Fig. ​(Fig.1).1). Two specific kinases catalyze the further phosphorylation of 5FUMP, eventually converting it to fluorouridine triphosphate. Fluorouridine triphosphate in turn gets incorporated into RNA, which causes miscoding, leading to the inhibition of fungal protein synthesis (Fig. ​(Fig.1).1). As a secondary method of inhibition, 5FUMP inhibits thymidylate synthetase (Fig. ​(Fig.1),1), leading to the depletion of dTTP and the misincorporation of dUTP into newly synthesized DNA, causing irreversible DNA damage and cell cycle arrest (14, 31, 41).Open in a separate windowFIG. 1.Metabolic pathway and mode of action of 5FC in Candida yeasts. 5FC and 5FU are transported into the cell by cell membrane-associated cytosine-purine permeases. In Candida spp., these are encoded by two genes that display amino acid sequence homology with the FCY2 gene of S. cerevisiae (13). Upon entry into the cell, 5FC is then deaminated to 5FU by Fca1p, encoded by FCA1. 5FU is then phosphorylated by UPRT, encoded by FUR1, yielding 5FUMP. 5FUMP inhibits thymidylate synthetase, which leads to thymidine depletion in the cell and which ultimately interrupts DNA synthesis. 5FUMP is also metabolized by two kinases, yielding fluorouridine diphosphate (5FUDP) and, subsequently, fluorouridine triphosphate (5FUTP), the latter of which is incorporated into RNA in the place of UTP, which leads to miscoding and the inhibition of protein synthesis.In haploid C. lusitaniae isolates, a missense T26C nucleotide mutation in the FCY1 gene has been reported in four clinical isolates demonstrating 5FC resistance, although 5FC and 5FC-fluconazole cross-resistance has more commonly been attributed to defects in the purine cytosine permease-encoded FCY2 gene in this species (10). In C. albicans, resistance to 5FC is mediated by a reduction in the activity of either the Fca1p encoded by FCA1 or the UPRT encoded by FUR1 (13, 31, 43). Two different research groups reported that in the majority of 5FC-resistant C. albicans isolates, resistance is associated with a homozygous single amino acid substitution, Arg101Cys, in UPRT (7, 13). However, other 5FC-resistant C. albicans isolates lack this substitution (13). One such isolate (5FC MIC, >64 μg/ml) was reported to contain a homozygous Gly28Asp substitution in the cytosine deaminase gene, and a Ser29Leu amino acid substitution was also observed in the same gene of another C. albicans isolate displaying intermediate 5FC resistance (5FC MIC, 4 μg/ml) (13). In C. dubliniensis, the DNA sequences of the FUR1 genes encoding the UPRTs of four 5FC-resistant and five 5FC-susceptible isolates from the Middle East were determined previously, and while several single nucleotide polymorphisms (SNPs) were identified, no amino acid substitutions were observed between the isolates (4).The purpose of the present study was to investigate the role of Fca1p in C. dubliniensis clade-specific 5FC resistance by the use of broth microdilution assays with 5FC and 5FU, analysis of DNA and amino acid sequences, and analysis of CdFCA1 expression. A tetracycline-inducible expression plasmid was used to incorporate the CdFCA1 gene from a 5FC-susceptible isolate (hereafter called CdFCA1^s) into the ADH1 locus of a 5FC-resistant isolate and the CdFCA1 gene from a 5FC-resistant isolate (hereafter called CdFCA1^r) into the ADH1 locus of a 5FC-susceptible isolate. These strains were used to determine if 5FC susceptibility or resistance could be induced in isolates upon the acquisition and expression of the respective CdFCA1 gene.     

Mutational Analysis of Flucytosine Resistance in Candida glabrata

The antifungal flucytosine (5-fluorocytosine [5FC]) is a prodrug metabolized to its toxic form, 5-fluorouracil (5FU), only by organisms expressing cytosine deaminase. One such organism is Candida glabrata, which has emerged as the second most common agent of bloodstream and mucosal candidiasis. This emergence has been attributed to the high rate at which C. glabrata develops resistance to azole antifungals. As an oral agent, 5FC represents an attractive alternative or complement to azoles; however, the frequency of 5FC resistance mutations and the mechanisms by which these mutations confer resistance have been explored only minimally. On RPMI 1640 medium containing 1 μg/ml 5FC (32-fold above the MIC, but less than 1/10 of typical serum levels), resistant mutants occurred at a relatively low frequency (2 × 10⁻⁷). Three of six mutants characterized were 5FU cross-resistant, suggesting a mutation downstream of the Fcy1 gene (cytosine deaminase), which was confirmed by sequence analysis of the Fur1 gene (uracil phosphoribosyl transferase). The remaining three mutants had Fcy1 mutations. To ascertain the effects of 5FC resistance mutations on enzyme function, mutants were isolated in ura3 strains. Three of seven mutants harbored Fcy1 mutations and failed to grow in uridine-free, cytosine-supplemented medium, consistent with inactive Fcy1. The remainder grew in this medium and had wild-type Fcy1; further analysis revealed these to be mutated in the Fcy2L homolog of S. cerevisiae Fcy2 (purine-cytosine transporter). Based on this analysis, we characterized three 5FC-resistant clinical isolates, and mutations were identified in Fur1 and Fcy1. These data provide a framework for understanding 5FC resistance in C. glabrata and potentially in other fungal pathogens.Candida glabrata has emerged in recent years as the second most common agent of mucosal and invasive candidiasis (16, 17, 20, 25). This emergence can be attributed largely to the intrinsically low susceptibility of C. glabrata to azole antifungals and its high capacity for acquired azole resistance. Azoles such as fluconazole, introduced in 1990, are used widely due to their low toxicity, availability in both oral and intravenous formulations, and excellent activity versus most other yeasts, including Candida albicans. With one exception, the remaining classes of antifungals are deficient in one or more of these properties; specifically, polyenes such as amphotericin B are toxic, echinocandins such as caspofungin can only be administered intravenously, and allylamines such as terbinafine lack anticandidal activity. The exception is the pyrimidine analog flucytosine (5-fluorocytosine [5FC]), which represents an attractive alternative or complement to azoles, with excellent activity against most C. glabrata isolates (MIC₉₀ of ≤0.12 μg/ml) (21) and the capacity for both oral and intravenous administration (although the latter formulations are not currently available in the United States). 5FC is also well tolerated when moderately dosed (e.g., serum level of 25 μg/ml) (29); however, higher, potentially toxic doses are often used in attempts to counter resistance (see below) or to broaden the spectrum of activity to fungi with intrinsically low susceptibility, such as Cryptococcus spp. (MIC₉₀ = 2 to 16 μg/ml) (22, 24).5FC is unique among antifungals in being a prodrug and in targeting a nonessential salvage pathway (Fig. ​(Fig.1).1). Studies of susceptible fungi, primarily the genetic model Saccharomyces cerevisiae, have shown that 5FC is taken up by one or more cytosine permeases (the most relevant is encoded by FCY2) (19, 32) and modified to 5-fluorouracil (5FU) by cytosine deaminase (encoded by FCY1 [also known as FCA1 in C. albicans]) (3). Subsequent modifications to 5-fluoro-UMP by uracil phosphoribosyl transferase (UPRT; encoded by FUR1) (11, 12) and to 5-fluoro-dUMP ultimately result in the disruption of protein and DNA synthesis. The absence of cytosine deaminase in mammalian cells provides the basis for selective toxicity. 5FU, in contrast, is highly toxic, and indeed, the conversion of 5FC to 5FU by gut bacteria may be largely responsible for 5FC toxicity (29).Open in a separate windowFIG. 1.Salvage pathway for cytosine (or 5FC) uptake and conversion to UMP (or 5-fluoro-UMP) in yeast (and in the case of 5FC, the downstream consequences on RNA, DNA, and protein synthesis). Also shown (in abbreviated form) are the alternative pathways for UMP production via the de novo pathway or uridine uptake.High rates of acquired resistance during monotherapy are considered a major limitation of 5FC therapy and are part of the rationale for the use of high, potentially toxic doses (29). However, these rates may differ significantly between the haploid species C. glabrata and diploid Candida species such as C. albicans and Candida tropicalis. In a large global survey, most isolates of these three species exhibited 5FC MICs of ≤0.25 μg/ml, and using MICs of ≥8 μg/ml as a breakpoint, only 1% of C. glabrata isolates were 5FC resistant (21). For C. albicans and C. tropicalis, the rate of resistant isolates increased, to 3 and 8%, respectively. Moreover, in a subset of C. albicans isolates (serotype B), the majority of isolates exhibit intermediate susceptibility or resistance (24). The predominant mechanism behind this was elucidated by Dodgson et al. (2) and Hope et al. (7). C. albicans isolates can be grouped genotypically into five major clades, and intermediate susceptibility and resistance strongly correlate with clade 1 (23). Dodgson et al. identified the mutation Arg101Cys (R101C) in Fur1, which is present in heterozygous form in intermediate isolates and in homozygous form in resistant isolates. The analogous mutation R99S (formerly labeled R134S based on an erroneous start site) was previously identified in an S. cerevisiae laboratory mutant (12). Thus, 5FC resistance results from a relatively high-frequency mitotic gene conversion event, as originally envisioned by Whelan (33), rather than requiring a much less frequent point mutation. Hope et al. confirmed these findings and, furthermore, identified specific mutations in Fca1 (G28D and S29L) responsible for 5FC resistance or intermediate susceptibility. The basis for 5FC resistance has also been explored in Candida dubliniensis (where it was associated with the Fca1 S29L mutation) (18), Candida lusitaniae (Fcy2 truncation or Fcy1 M9T mutation) (5), and most recently, C. glabrata (Fur1 G190D mutation, Fcy1 W148R mutation, and Fcy2 G246S mutation) (1, 28).The mechanism by which these mutations confer resistance can be inferred from previous work with S. cerevisiae. Specifically, null mutants (including disruptants) of the genes encoding Fcy1, Fur1, and Fcy2 confer 5FC resistance (3, 9, 11, 19). This is feasible because the salvage pathway employing these enzymes is nonessential, i.e., UMP can also be synthesized from exogenously acquired uridine or de novo from l-glutamine (Fig. ​(Fig.1).1). An understanding of 5FC resistance mechanisms in pathogenic yeast would facilitate rapid, molecular technology-based detection and may also suggest novel ways to reduce or reverse resistance. Toward this goal, we present here an analysis of 5FC resistance mechanisms in C. glabrata. In laboratory mutants, resistance occurred at a moderately low frequency, and mutations were distributed between Fcy1, Fur1, and the Fcy2 paralog Fcy2L. Mutations in Fcy1, Fur1, and the Fcy2 paralog Fcy2J were subsequently identified in a small set of clinical isolates exhibiting reduced 5FC susceptibility.     

Clinical Pharmacokinetics of Oral Controlled-Release 5-Fluorocytosine

5-Fluorocytosine (5FC) is an oral antifungal that is currently used in combination with amphotericin B to treat Cryptococcus neoformans meningoencephalitis. The oral dosing of 5FC could be optimized by the use of a controlled-release (CR) formulation. The objective of the current study was to develop two prototype 5FC-CR formulations and evaluate the single-dose (1,500-mg) serum pharmacokinetic profiles of those formulations relative to the profile of the commercially available, immediate-release 5FC product (Ancobon) by the use of a phase 1, open-label, randomized, three-phase, crossover pharmacokinetic study design. Hydroxypropyl methylcellulose was utilized as the rate-controlling matrix to compound the 5FC-CR tablets. The two prototype 5FC-CR formulations demonstrated 80% release at 13.0 and 18.4 h, respectively, whereas the immediate-release product demonstrated 80% release at 0.28 h, as determined in vitro by the United States Pharmacopeia apparatus 2 dissolution method. Five subjects completed all three phases of the study without any adverse events. The mean maximum concentration, the area under the curve from time zero to 24 h, and the area under the curve from time zero to infinity were approximately 50% lower (P < 0.01) with the 5FC-CR formulations than with the immediate-release 5FC product. However, no statistically significant differences in the minimum concentrations at 24 h were noted between the formulations. The gastric absorption profile of 5FC-CR was well predicted by in vitro dissolution. Future exploration of a gastroretentive 5FC-CR formulation could overcome the marked lack of bioequivalence observed in the present study.Cryptococcus neoformans is an opportunistic fungal pathogen that is associated with significant morbidity and mortality. A 27% mortality rate has been associated with Cryptococcus neoformans meningoencephalitis (CNME) among AIDS patients in South Africa (15). An induction antifungal regimen of amphotericin B (AMB) and 5-fluorocytosine (5FC) for 2 weeks, followed by long-term fluconazole (FLZ) maintenance therapy, is considered the standard of care for AIDS patients with CNME (20). While it is effective, the use of induction therapy has been too complex for implementation in developing countries (11). The use of FLZ monotherapy in AIDS patients with CNME has been utilized but is associated with treatment failure and the emergence of resistance (3, 18). Hence, access to a simple, easy-to-administer combination oral antifungal regimen could improve CNME-related outcomes in countries without sufficient resources. The use of oral 5FC and FLZ combination regimens have demonstrated promising clinical results (4, 8, 10, 14, 16).However, currently recommended 5FC dosing regimens may not be optimal on the basis of pharmacokinetic (PK) and pharmacodynamic (PD) principles (7). For example, administration of 5FC at 25 mg/kg of body weight by mouth every 6 h yields steady-state maximum plasma concentrations (C_maxs) of 60 to 80 μg/ml and minimum plasma concentrations (C_mins) of 5 to 20 μg/ml in patients with normal renal function (26). The PK-PD index predictive of the effects of 5FC against C. neoformans is most likely the time that the concentration remains above the MIC (T > MIC) (1, 13). The MIC₉₀ of C. neoformans var. grubii is approximately 8 μg/ml on the basis of the findings of a survey of nearly 2,000 isolates collected worldwide (19). Given that 5FC has a low level (4%) of protein binding and concentrations in cerebrospinal fluid (CSF) that are 80% of the values in serum, a serum C_min above 10 μg/ml should theoretically achieve a 100% T > MIC in CSF (26). An oral controlled-release (CR) 5FC (5FC-CR) formulation could be used to maintain a steady serum concentration above 10 μg/ml. Moreover, the use of lower 5FC doses could reduce the nausea, vomiting, and bone marrow suppression that have been associated with high plasma 5FC concentrations (26). The combined use of 5FC-CR and FLZ could also simplify oral administration (once daily) and improve outcomes among AIDS patients with CNME in sub-Saharan Africa.This report outlines the development of two model CR formulations of 5FC. The PK profile of a single dose of immediate-release (IR) 5FC (Ancobon) capsules was compared to the PK profiles of two 5FC-CR formulations in a cohort of five subjects by the use of a three-phase crossover pharmacokinetic study design. The objectives of this pilot study were to (i) determine the feasibility of creating a 5FC-CR formulation, given its relatively low molecular weight and hydrophilic chemical structure and the high dose that is required and (ii) compare the serum PK profile of a single dose of 5FC-CR to that of the commercially available IR product in healthy volunteers.(This work was presented in part at the 2007 American Association of Pharmaceutical Scientists Annual Meeting, San Diego, CA, 11 to 15 November 2007, and at the 2008 Controlled Release Society Annual Meeting, New York City, NY, 12 to 16 July 2008.)     

Environmentally friendly approach to recover vanadium and tungsten from spent SCR catalyst leach liquors using Aliquat 336

This research paper deals with an environmentally friendly approach for the treatment of spent selective catalytic reduction (SCR) catalyst. To recover vanadium (V) and tungsten (W) from spent SCR catalyst, leach liquors from hydrometallurgical processing were utilized to develop a proper methodology for extraction and possible separation of vanadium and tungsten from each other. This study investigated the solvent extraction (also called liquid–liquid extraction) of vanadium and tungsten utilizing the alkaline roasted leached solution containing approximately ∼7 g L⁻¹ of tungsten and ∼0.7 g L⁻¹ of vanadium. The commercial extractant, N-methyl-N,N,N-tri-octyl-ammonium chloride [R₃NCH₃]⁺Cl⁻ (commercial name Aliquat 336), was dissolved in Exxsol™ D80 (diluent) system and adopted in this research. Solvent extraction studies were performed to determine the following experimental parameters: equilibrium pH, extractant concentration, diluent influence, chloride ion concentration, temperature, and stripping reagent concentration, which were systematically scanned to ascertain the optimum conditions for quantitative extraction of both title metals. An anion exchange mechanism was proposed using the quaternary ammonium chloride solvent reagent after slope analysis. Excess supplement of chloride proved to have adverse effects, further supporting the extraction mechanism. Thermodynamics results show positive values for enthalpy (ΔH) for vanadium and tungsten, favoring the endothermic nature of the extraction reaction towards the uptake of either metal. McCabe–Thiele plots for extraction were constructed, suggesting 2 and 3 stages for vanadium and tungsten extraction, respectively, at the aqueous (A) to organic (O) phase ratio of 7 : 1, ensuring more than 99.9% and 7-fold enrichment of both title metals. The stripping trend follows the order: (NaOH + NaCl) > (NaOH + NaNO₃) > NaOH > NaNO₃ > NaCl. Stripping isotherm followed by stripping counter-current (CCS) study was carried out for quantitative stripping of the metals.

A complete extraction and stripping process to obtain enriched vanadium and tungsten concentrate from spent SCR catalyst leach liquor.     

The enhancement of treatment capacity and the performance of phytoremediation system by fed batch and periodic harvesting

Floating macrophyte phytoremediation could be the most relevant solution to the ever-increasing finfish farm pond effluent worldwide. However, the information of Spirodela polyrhiza monoculture system in fed batch mode, with periodic harvesting and increased macrophyte density is limited. In this study, the effect of fed batch and periodic harvesting on the treatment capacity and performance of the S. polyrhiza monoculture system (with increased the macrophyte density) in fish farm wastewater were evaluated. Results showed that the system with fed batch and harvesting could treat a greater volume of wastewater, remove a higher amount of pollutants in terms of ammonia (NH₃–N), phosphate (PO₄³⁻), total suspended solids (TSS) and chemical oxygen demand (COD), while meeting the effluent limits. The system with S. polyrhiza macrophyte density of 11.67 g fresh weight (FW) per L wastewater was able to decrease nitrate (NO₃⁻–N) and nitrite (NO₂⁻–N) to an undetected level. This study suggested that the S. polyrhiza monoculture system with fed batch, optimal harvesting and frequent sediment removal is feasible and effective in treating the fish farm wastewater, and produces biomass with superior protein content for fish feed supplement and poultry diet. The obtained data provided insights into the system reliability in wastewater treatment and ways of improvement for the system. The treated wastewater could achieve exceptional quality with minimal toxicity before discharge to receiving waters, and potentially be reused for water flow recharge, aquaculture and irrigation purposes, minimizing the pollution and ecological impacts.

Spirodela polyrhiza FBH system had 150% more treatment capacity, up to 5.2× higher removal in NH₃–N, PO₄³⁻, TSS and COD.     

A novel switchable-hydrophilicity,natural deep eutectic solvent (NaDES)-based system for bio-safe biorefinery

A switchable-hydrophilicity solvent system, consisting of a fatty acid-based natural deep eutectic solvent (NaDES), complemented by a bio-friendly dilute amine solution, has been introduced. The potential of the most benign switchable solvent system has been characterised in microalgae biorefining according to the recently proposed ‘Circular Extraction’ scheme.

A switchable-hydrophilicity solvent system, consisting of a fatty acid-based natural deep eutectic solvent (NaDES), complemented by a bio-friendly dilute amine solution, has been introduced and used for extractions from microalgal biomass.

“Biorefining”, “market value” and “destination-neutrality” are almost inseparable concepts. “Biorefining”, the operation consisting of separating the individual compounds of biomass, such as microalgae, aims at existing or new market segments, thus implying the concept of “market value”, the amount of money that can be obtained from a given assembly of compounds. However, certain compounds, or assemblies thereof, may have multiple markets (e.g., biomaterials, detergents, food, feed, pharma, biofuel, and fertilisers, to just name a few key ones), where similar assemblies are subjected to different regulations and may be accordingly, differently valued. The term “destination-neutrality” denotes the possibility that a compound or an assembly may meet the restrictions entailed by the regulations of different markets; this is a key concept for creating maximum value for microalgal-derived products.Switchable hydrophilicity solvents (SHS) are a new class of solvents that are able to change their nature from hydrophobic to hydrophilic and vice versa.¹ So far, SHS systems have been created by biphasic systems composed of a hydrophobic liquid organic base and an aqueous layer. Upon addition of CO₂, the liquid base becomes protonated and the resulting bicarbonate salt is fully miscible with water, converting the entire mixture into a single phase. Common SHS functional groups include alkylated amidines or secondary and tertiary amines that act as liquid bases to deprotonate carbonic acid or hydrated CO₂.² Recently, Chen et al. described a switchable hydrophilicity system where the solvent is a fatty acid and the hydrophilic phase to make it switch is based on a dilute aqueous solution of a water-soluble amine.³ The switching mechanism, in this case, is different from that described above, in that the amine is able to create a complex with the fatty acid, so that the oily phase is entirely dissolved into the watery phase and the whole system becomes hydrophilic. Addition of CO₂ reprotonates the carboxylate anion, causing the hydrophobic carboxylic acid to phase separate from the aqueous phase. Switchable hydrophilicity solvents have been used in their hydrophobic form to extract hydrophobic solutes, such as oil from soybean flakes⁴ and microalgae,⁵ astaxanthin from microalgae,⁶ phenols from lignin-derived bio-oils⁷ and herbicides from water samples.⁸ In all of these studies the focus is limited to the extraction of only one compound or fraction from the biomass (or the liquid phase), the switching serving the purpose of separating that compound or fraction from the solvent. The SHS in the switched hydrophilic state is not used and must therefore be brought back to the initial hydrophobic condition prior to further use, so that the hydrophilic state of the SHS is necessary for the overall process but useless as far as extraction is concerned.In our previous work we discussed the possibility of increasing the extraction of microalgal biomass components by exploiting both the native form of the SHS and the hydrophilic form obtained after the switching process, thereby increasing the overall utility of both the algae and the solvent.⁹ We also showed the power of this approach in contributing to the biomass fractionation into the main classes of biologically-relevant substances, and the entailed opportunity for optimising this fractionation by adopting the “forward-mode” (carrying out the extraction first by using the hydrophobic form of the SHS, and then the hydrophilic form) or the “backward-mode” (carrying out the extraction first by using the hydrophilic form of the SHS, and then switching back to the hydrophobic form for solvent recovery and then extraction of hydrophobic components) that can be adopted for the overall solid–liquid extraction unit operation (Fig. 1). It should be noted that Fig. 1 refers only to conceptual facts and does not care about their time sequence. Square and circle mean “hydrophilic step” and “hydrophobic step”. Arrows tell the reader what goes in and out, without caring about time. The curved arrows going from the circle to the square and vice versa indicate that the hydrophobic phase becomes hydrophilic and vice versa.Open in a separate windowFig. 1Sequential two-stage extraction in forward (hydrophobic first, A) and backward (hydrophilic first, B) modes.In this work, a new SHS system with a substantial novelty is introduced in that it is based on a natural, deep eutectic solvent (NaDES) made of fatty acids and a weak amine water solution (Fig. 2). The NaDES which was adopted here is the mixture of octanoic acid and dodecanoic acid described by Florindo et al. and which the original inventors used only in its native state, as often done for NaDES systems.¹⁰ This NaDES is highly hydrophobic and exhibits a solidification temperature of 9 °C (compared to 16 °C and 43.8 °C of the individual acids), so that it is normally in the liquid state at ambient temperature. This system was complemented by a dilute (5%) aqueous solution of Jeffamine D-230, as described by Chen et al.³ It should be noted that, while a single fatty acid solvent system suffers from high solidification temperature, the fatty acid-derived NaDES is more favourable in this respect. In the following, we will call the fatty acid deep eutectic solvent simply NaDES-Y, and the associated Jeffamine D-230 solution “amine solution”, for brevity.Open in a separate windowFig. 2NaDES-Y switching system, from hydrophobic form to hydrophilic and vice versa.In the present work, we demonstrate that NaDES-Y: (1) can be made to switch, thus forming a hydrophilic phase; (2) can be made to switch back, with appropriate means, thus returning to the initial hydrophobic state; (3) can be used to solubilise triacylglycerides, and that these ones can be recovered upon switching the SHS to its hydrophilic form; (4) can be used to extract the biological fractions (proteins, carbohydrates and lipids) making up biomass. This latter part of the work was carried out according to the “Circular Extraction” paradigm⁹ in two subsequent steps and in both the “forward-mode” and “backward-mode”: (4.1) a synthetic matrix representing the microalgal biomass, made of selected proteins, lipids, carbohydrates, and water, was extracted by NaDES-Y and the extraction streams were characterised; (4.2) a sample of the adopted, wet microalgal biomass was extracted by NaDES-Y and the extraction streams were characterised.In the first part of the experiment, 5 mL of NaDES-Y were mixed with the amine solution, in the volume ratio 1 : 13, and thoroughly mixed, thus obtaining a single, hydrophilic phase and demonstrating that the NaDES-Y can be switched in situ.In the second part of the experiment, CO₂ was bubbled into the mixture, thus acidifying it, reprotonating the carboxylate anions of the NaDES-Y and causing a phase splitting between the NaDES-Y and the amine solution. We have ascertained that the hydrophobic phase that separates from this phase splitting is still the original NaDES-Y by checking that its solidification temperature is unchanged, thus demonstrating that the NaDES-Y can also be switched-back in situ. It should be noted that the reversal of the system to the initial split-phase state can be obtained not only by injecting CO₂ but also by acidification with strong acids, such as HCl, although the use of the latter would likely increase the overall environmental impact of the method and would not be reversible, e.g. upon flushing with a gas stream such as air.In order to test the suitability of this method for lipid solubilization and release, sunflower oil was used as a model system for triacylglycerols as in Jessop et al.¹¹ 5 mL of NaDES-Y and 1 mL of sunflower oil were mixed. A single phase was obtained. Upon adding the aqueous amine solution and mixing, a phase split was produced between a hydrophilic phase consisting of the water and ammonium carboxylate salts of the hydrophilic NaDES-Y, and a hydrophobic phase made by sunflower oil.Prior to the fourth part of the work, microalgal biomass of Scenedesmus dimorphus (UTEX 1237) was cultivated in our laboratory and then compositionally assessed. Carbohydrates and proteins were quantified colorimetrically with spectrophotometry; total carbohydrates were quantified by the Dubois assay¹² and total proteins were quantified by the Lowry assay.¹³A model matrix was prepared by blending starch (0.38 g), glucose (0.091 g), casein (0.177 g, adopted as a widely available hydrophobic protein), albumin (0.158 g, adopted as a widely available hydrophilic protein), sunflower oil (0.09 g adopted as a widely available triacylglycerides pool), soy lecithin (0.04 g adopted as a widely available phospholipids pool to represent cellular membranes) and water (0.75 g adopted to represent intrinsic and extrinsic water). The composition of this matrix was chosen to simulate the measured composition of S. dimorphus and the water content of “wet” microalgal biomass separated by centrifugation of the cultured suspension (20 minutes at 2600g). Subsequently, the extraction of the model matrix was carried out with NaDES-Y, in its native hydrophobic state. The model matrix (1.58 g) was thoroughly mixed with 30 mL of NaDES-Y and agitated for 24 h in the presence of glass beads. Then agitation was interrupted and any insoluble material was separated and stored for the second-stage extraction. The aqueous amine solution was added to the homogeneous NaDES-Y thus causing the phase split that expels oily fractions. The now-hydrophilic solvent was then collected and used to further treat the insoluble material that had been stored at the end of the first extraction stage, again by prolonged thorough mixing. Finally, any still insoluble material was removed and the liquid was switched back to its initial state. By following the two extraction stages described here, the complete “forward-mode”, dual-stage circular extraction was complete, and the extraction capability of the SHS was assessed by characterising both the supernatant streams and the solid residuals.In a separate experiment, the extraction order was reversed, thus following what was named the “backward-mode” circular extraction in our previous work.⁹ In this case the model matrix was first treated with NaDES-Y that had been already mixed with the amine solution, thus becoming hydrophilic, producing an aqueous liquid phase and an intermediate solid residue that was stored. After addition of CO₂ to the liquid phase, triggering a phase split, the supernatant (the hydrophobic NaDES-Y), was decanted from the aqueous phase that contained the extracted hydrophilic components from the model biomass mixture. The recovered NaDES-Y was then used to extract hydrophobic components from the intermediate solid residue, thus obtaining a second extract and a final insoluble residue. All streams were compositionally assessed as before, thus completely characterising the backward-mode dual-stage extraction in the synthetic matrix.All of the above extraction procedures, both in forward mode and in backwards mode, were also performed on the microalgal biomass rather than the synthetic matrix. First, however, we investigated the capability of the solvent to break the cell wall by agitating a microalgal cells-in-solvent suspension for 24 h. The very poor yield in a subsequent forward-mode extraction indicated that the mildly acidic pH (∼2) of the solvent was unable to cause cell rupture by itself, and that an additional cell disruption method would be necessary. Further extractions were performed in the presence of glass beads, which greatly improved the extraction of lipids, and moderately improved the extraction of proteins and carbohydrates. A further provision to boost the extraction of the lagging fractions was the application of microwaves (90 s at 300 W in a household oven). However, when carrying out the “forward-mode” extraction, we realised that the combined effect of microwaving (which implies heating dipoles such as water) in the presence of an acidic pH (imparted by the fatty acids) caused an extensive degradation of chlorophyll (to pheophytin), a well-known undesired outcome of misperformed sterilisation processes of vegetables in the food industry. The solution, here, came from the very solution adopted in vegetables sterilisation, that is inducing a mild alkalinity in the food mass that must be sterilised. However, in circular extraction, it is not necessary to actually perform any alkalinisation, because it is sufficient to adopt the “backward-mode” instead of the forward mode extraction. Indeed, achieved the desired outcome of higher yields without extensive degradation of chlorophyll.The quantitative results of parts 4.1 and 4.2 are reported in Fig. 3 and ​and44 (model system, forward- and backward-mode), 5, 6 and 7 (microalgal biomass, various extraction implementations in forward- and backward-mode).Open in a separate windowFig. 3Fractional extraction efficiencies on model system extracted in forward-mode.Open in a separate windowFig. 4Fractional extraction efficiencies on model system extracted in backward-mode.The reported results should be interpreted as follows: during the forward-mode extraction (Fig. 2), 41% of the carbohydrates contained by the model matrix were dissolved by NaDES-Y, which has a hydrophobic character. Switched NaDES-Y, which has a hydrophilic character, managed to extract a further 26% of the initial carbohydrates that had remained in the matrix residue after the first stage of the forward-mode extraction. The total carbohydrates extraction reached therefore 67%. It should be noted that the total extraction is split between two streams; therefore, their recovery should be performed through both the hydrophobic-to-hydrophilic switching and the hydrophilic-to-hydrophobic switching. The backward-mode extraction (Fig. 2) has different extraction yields, but the same concept holds.Synthetic matrix extractions results (Fig. 3 and ​and4)4) show that, as far as total extraction is concerned, separate consideration may be made for proteins, carbohydrates and neutral lipids, and their distribution in the two subsequent extract streams obtained. Neutral lipid extraction is almost quantitative whatever orientation (forward or backward) of the operation is adopted and protein extraction is nearly quantitative, with a slight preference for backward rather than forward orientation (99% vs. 93). In the case of carbohydrates, a more marked difference between forward and backward orientation is observed (66% vs. 86%), again with an advantage of the backward mode over the forward mode. From the point of view of fraction distribution between extracts, neutral lipids are exclusively obtained from the hydrophobic extract and are absent in the hydrophilic extract, whatever the orientation of the operation.Protein extraction, on the other hand, appears to be split between the two subsequently staged extractions in the opposite way. The extraction occurring in the first place picks up a large fraction of the proteins (85% if it is hydrophobic, 88% if it is hydrophilic), leaving a much smaller fraction to be accomplished by the extraction performed subsequently (8% if it is the hydrophilic follow-up of a forward-mode extraction, 11% if it is the hydrophobic follow-up of a backward-mode extraction). Carbohydrates extraction shows a behaviour that is similar, albeit less marked, to that of proteins. The extraction occurring first picks up a comparatively larger fraction of the proteins originally contained in the matrix (41% if it is hydrophobic, 49% if it is hydrophilic), leaving a somewhat smaller fraction for the extraction coming later (26% if it is the hydrophilic follow-up of a forward-mode extraction, 38% if it is the hydrophobic follow-up of a backward-mode extraction).While the result observed for neutral lipids was expected, explanation for the behaviour of the proteins is less intrinsically clear. Proteins are equally distributed between hydrophilic- and hydrophobic-character in the synthetic matrix. However, well above the available amount of protein matching the type of solvent (hydrophilic vs. hydrophobic) is extracted in the extraction stage coming first. This might be due to the formation of micellar systems created by the phospholipids that were added to represent cell membranes and eased the extraction, pretty much as it occurs in micelle-assisted protein recovery techniques;¹⁴ indeed, the rough solubility of casein (representing hydrophobic proteins) in native (hydrophobic) NaDES-Y is 0.24 g L⁻¹, while the calculated casein concentration in our hydrophobic extract (first stage of the forward-mode extraction of the synthetic matrix) is 4.60 g L⁻¹; the rough solubility of albumin (representing hydrophilic proteins) in native NaDES-Y is 0.22 g L⁻¹, while the calculated albumin concentration in our hydrophobic extract is 4.00 g L⁻¹. Analogously, the rough solubility of casein in the hydrophilic form of NaDES-Y is 0.06 g L⁻¹, while the calculated casein concentration in our hydrophilic extract (from the first stage of the backward-mode extraction of the synthetic matrix) is 4.35 g L⁻¹; the rough solubility of albumin in hydrophobic-to-hydrophilic-switched NaDES-Y is 0.52 g L⁻¹, while the calculated albumin concentration in our hydrophilic extract is 4.45 g L⁻¹. Carbohydrates are not only more extensively extracted in backward-mode in the term of the total amount, but they are also dissolved more in each partial step of it (both the first and the second step show a +10% increase).The results of part 4.2 of the work show that the method of cell disruption has a strong effect on the yield of extract. As mentioned above, barely agitating a microalgal suspension in either hydrophobic or pre-switched NADES-Y does not succeed in ensuring a significant extraction yield most likely due to the unbroken cell wall hindrance and was not investigated further (the extraction efficiency resulted to be 2.0% ± 2.6% for proteins, 5.1% ± 4.4% for carbohydrates, with only traces of neutral lipids). When bead beating was added, a significant improvement to lipid yield was recorded during the hydrophobic step of the forward-mode extraction (88%) (Fig. 5), while protein and carbohydrate extraction was essentially unchanged. However, during the subsequent hydrophilic stage of the forward-mode extraction, a further 32% and 25% of the microalgal proteins and carbohydrates, respectively, were picked up from the microalgal matrix, thus leading to an overall extraction of 36% of the original proteins and 33% of the original carbohydrates. When reversing the extraction (backward-mode) (Fig. 6), 17% and 8% of the original proteins and carbohydrates, respectively, were dissolved during the hydrophilic stage; after hydrophilic-to-hydrophobic switching the solvent, a further 28% (proteins) and 17% (carbohydrates) extraction was possible, thus attaining an overall extraction ratio of 45% and 25%, respectively, of the original proteins and carbohydrates of the microalgal matrix. Lipids were extracted exclusively in the hydrophobic stage, and total extraction did not vary appreciably between the forward-mode and the backward-mode conduite of the dual-stage extraction. It is therefore apparent that beads beating ensures a fair yield in lipids, while proteins and carbohydrate extraction is promoted to a lesser degree. The second cell disruption technique tested, microwave-assisted extraction, could only be applied to the hydrophilic phase and, although it could also have been limited to the hydrophilic stage of the forward-mode extraction, in this study it was limited to the (hydrophilic) first stage of the backward-mode extraction. Results (Fig. 7, “microwave-assisted microalgal suspension extraction”) show a clear improvement in extractability of proteins during the first (hydrophilic) stage of the extraction (54% vs. 17% in the first stage of the bead-beaten extraction), while protein extraction in the second stage (hydrophobic, un-microwaved) became less efficient (12% vs. 28% in the first stage of the bead-beaten extraction). Overall protein extraction ratio, however, jumped from 45% to 72%. Carbohydrates resembled the proteins behaviour in a mirrored way: their extractability during the first (hydrophilic) stage of the extraction was only slightly promoted by the microwave treatment (18% vs. 8% in the first stage of the bead-beaten extraction), while in the second stage (hydrophobic, un-microwaved) their extraction was boosted (41% vs. 17% in the second stage of the bead-beaten extraction). Overall carbohydrate extraction, therefore, was significantly promoted, from 25% to 59%.Open in a separate windowFig. 5Fractional extraction efficiencies on bead beating-assisted microalgal suspension extraction in forward-mode.Open in a separate windowFig. 6Fractional extraction efficiencies on bead beating-assisted microalgal suspension extraction in backward-mode.Open in a separate windowFig. 7Fractional extraction efficiencies on microwave-assisted microalgal suspension extraction in backward-mode.In combination with a cell wall rupturing technique, therefore, the anticipated extraction potential toward biologic fractions recorded during synthetic matrix extraction experiments was well confirmed on microalgal matrix for lipids, while a 20–60% lower extraction was recorded for proteins and carbohydrates, most likely due to residual diffusional hindrances and inter-fraction cross-link effects which are the fundamental (non compositional) difference between the synthetic and the natural matrix.Compared to the case study reported in the original ‘Circular Extraction’ article, in the present work extraction ratios are broadly comparable in the cases where NaDES-Y extraction is assisted by some means which is capable of breaking cell walls. However, depending on the type of cell disruption technique deployed, the results may be inferior, comparable, or superior to those obtained with DMCHA. Thus, bead beating-assisted extraction comes close to DMCHA extraction for proteins (36% vs. 41%) and lipids (88% vs. 96%) but is less efficient with carbohydrates (33% vs. 51%) in forward-mode extraction. In backward-mode extraction NaDES-Y, again, comes close to DMCHA efficiency for proteins (45% vs. 52%) and lipids (89% vs. 93%), while carbohydrate extracting power is half that of DMCHA (25% vs. 50%). If microwaved extraction is used the extraction with NaDES-Y improves significantly, as noted before, and thus NaDES-Y ranks as the best extracting medium for proteins (72% vs. 52%) and carbohydrates (59.3% vs. 51.4%) and comes very close to DMCHA efficiency for lipids (93% vs. 96%).From a biorefinery application perspective, adopting the bead beaten-assisted forward-mode extraction may warrant extracting lipids (88%) during the hydrophobic stage while proteins and carbohydrates extraction task is covered by the hydrophilic stage. On the other hand, by adopting the microwave-assisted extraction backward-mode extraction, during the hydrophilic stage NaDES-Y is able to extract more proteins than DMCHA (54% vs. 50%), with less contamination by co-extracted carbohydrates (18% instead of 47%), while the subsequent hydrophobic stage can extract more carbohydrates than DMCHA-based would (41% vs. 4%) in the same stage. While it may be observed that lipids would be co-extracted, this would not actually cause any recovery problem, given that the subsequent hydrophobic-to-hydrophilic form switch bring their separation about as shown in part 3 of the present results.     

Antifungal Combinations against Simulated Candida albicans Endocardial Vegetations

The in vitro effects of flucytosine (5FC), liposomal amphotericin B (L-AmB), and micafungin (Mica) combinations against two Candida albicans strains that simulated 24-hour-old endocardial vegetations were studied. Mica was superior to 5FC or L-AmB, and the 5FC-L-AmB-Mica combination was superior to all other treatments for one strain but no different from the dual combination of L-AmB-Mica for the other strain.Candida species can adhere to cell surfaces and form a three-dimensional community of microorganisms encased in an exopolymeric matrix known as biofilm (5). Biofilm can form on native and prosthetic heart valves to cause infective endocarditis. Candida albicans is the most common fungus linked to the development of infective endocarditis and is associated with a mortality rate in excess of 50% (6). Candida albicans organisms within biofilm have decreased cell membrane ergosterol content, have reduced expression of ergosterol biosynthetic genes, express higher levels of genes involved in amino acid and nucleotide metabolism, and upregulate efflux pumps (5, 9). These alterations may explain the poor activity of antifungals such as fluconazole and amphotericin that target ergosterol. Unlike these agents, the echinocandins target the cell wall of Candida species through inhibition of β-1,3-glucan synthase. A growing body of evidence suggests that echinocandins are active against Candida albicans biofilm (1, 4, 7, 8, 11, 13-15). However, cell wall integrity pathways and glucan-associated changes can also occur, which could limit echinocandin activity against Candida biofilm (10). As a consequence, combination antifungal therapy remains a rational approach to treatment of Candida endocarditis while possibly limiting emergence of resistance.The effectiveness of combination antifungal therapy for Candida endocarditis is difficult to assess clinically. Animal models provide valuable data but are not always necessary for initial assessment. Replication of antifungal pharmacokinetics to mimic the human profile cannot be easily accomplished in animal models. Also, evaluation of the interaction of antifungals requires large sample sizes and can lead to unnecessary animal testing with limited information gain. In contrast, in vitro models provide an alternative, more rapid, and controlled environment for the assessment of antifungal combinations. My coworkers and I previously determined the activity of flucytosine (5FC), micafungin (Mica), and voriconazole as single agents and in combinations against Candida species by using an in vitro model of infective endocarditis (11). This work demonstrated the poor activity of voriconazole and the superior activity of 5FC and Mica against developing Candida biofilm. As a consequence, the present study was performed to test the interaction of 5FC, Mica, and liposomal amphotericin B (L-AmB) against mature (24-hour-old) C. albicans human platelet fibrin clots that simulated endocardial vegetations (SEVs).Two C. albicans isolates, ATCC 14053 (CA1) and a clinical isolate (CA2) obtained from a septic patient with probable endocarditis collected from the University of New Mexico Hospital, were used for these experiments. Antifungal susceptibility testing was performed using the CLSI M27-A2 broth microdilution methodology for 5FC (Sigma, St. Louis, MO) and Mica (Astellas Pharma USA, Deerfield, IL). Both isolates were susceptible to 5FC with a MIC of 0.125 μg/ml. Amphotericin B (AmB) MICs were determined using the Etest methodology and were 0.25 μg/ml (CA1) and 1.0 μg/ml (CA2). The Mica MICs were 0.015 μg/ml (CA1) and 0.030 μg/ml (CA2). The C. albicans strains used in this study were selected because they reflected MIC₅₀s and MIC₉₀s of Mica from global surveillance data (12). A single colony of C. albicans obtained from a 24-h culture on Sabouraud dextrose agar (Cole-Parmer, Vernon Hills, IL) was grown in yeast nitrogen base medium (Difco Laboratories, Detroit, MI) supplemented with 2% dextrose at 27°C for 24 h. Infected fibrin clots were prepared as previously described but at a starting inoculum of ∼10⁴ CFU/g (11). The smaller inoculum permitted growth of C. albicans to ∼10⁶ CFU/g at 24 h just prior to exposure to antifungals.A one-compartment infection model (250 ml) was utilized in duplicate as previously described (11). The experiments were conducted over 72 h, which included a 24-h biofilm development phase followed by a 48-h treatment phase. Fungal burden was determined at six time points: −24 (baseline), 0 (treatment initiation), 8, 24, 32, and 48 h. At every time point, two vegetations were removed from each model, weighed, placed in a 10-ml sterile test tube prefilled with normal saline, and homogenized. After serial dilution, a 20-μl aliquot was plated in triplicate onto Sabouraud dextrose agar (Cole-Parmer, Vernon Hills, IL) and incubated for 24 h at 35°C and the colonies were counted visually. The same model apparatus using yeast nitrogen base-2% dextrose as the reservoir medium and SEVs was used to compare the single, dual, and triple combination activities of the aforementioned agents. All antifungal agents were administered as bolus doses 24 h after retention of SEVs in the model to permit biofilm formation. Antifungals were administered to simulate (70-kg patient) doses of 37.5 mg/kg of body weight every 12 h (5FC), 5 mg/kg every 24 h (L-AmB), and 150 mg every 24 h (Mica). Peristaltic pumps were activated to mimic an elimination half-life of 6 h for 5FC and 12 h for L-AmB and Mica. Two fibrin platelet clots were removed from each model at each time point and handled as described above given that antifungal carryover was not demonstrated. The log₁₀ CFU/g over time (0 to 48 h) was also compared between the different regimens tested and growth controls. The maximum rate of kill (K_max), area under the rate of kill curve (AURKC), and area between the treatment and control time-kill curves (ABTKC) were calculated (11). The seven treatment groups were compared using one-way analysis of variance with post hoc comparisons using Bonferroni correction for multiple comparisons of significance. Consequently, a P value of <0.007 was considered significant.Time-kill curves for both C. albicans isolates are illustrated in Fig. ​Fig.11 by isolate. The use of Mica alone was associated with a significant reduction in fungal burden over 48 h compared to either 5FC or L-AmB for both isolates. The mean ± standard deviation (SD) change in log₁₀ CFU/g between time zero and 48 h was 1.05 ± 0.55, 2.05 ± 0.77, and −2.98 ± 0.35 for 5FC, L-AmB, and Mica, respectively (P < 0.001). The effect of L-AmB was greater against isolate CA1 than against CA2, which was consistent with the lower AmB MIC against CA1. However, no significant reduction of fungal burden was noted for either 5FC or L-AmB over time regardless of isolate. A summary of time-kill curve parameters is included in Tables ​Tables11 and ​and22 based on isolate. The use of Mica alone was superior to 5FC and L-AmB based on ABTKC, AURKC, and K_max against both isolates. The triple combination of 5FC-L-AmB-Mica was superior (<0.007) to all other treatments based on ABTKC and K_max for isolate CA1. In contrast, the dual combination of L-AmB-Mica was superior (P < 0.007) to all other treatments except the triple combination of 5FC-L-AmB-Mica against CA2. Despite, these superior effects of combination therapy, no synergistic combinations were identified for the triple combination based on regression. The mean interaction (95% confidence interval) coefficients for this triple combination were −12.8 (−47.5, 21.8) and 16.7 (−13.3, 46.8), indicating indifference against CA1 and CA2, respectively.Open in a separate windowFIG. 1.Effects of 5FC, L-AmB, and Mica on mean ± SD log₁₀ CFU/g of Candida albicans (CA1 and CA2) SEV over time. Antifungals were introduced into the model at time zero to permit a 24-h biofilm maturity time.

TABLE 1.

Comparison of time-kill curve parameters of 5FC, L-AmB, and Mica combinations against C. albicans isolate CA1

Amphoteric starch derivatives as reusable flocculant for heavy-metal removal

A pH-responsive amphoteric starch derivative (PRAS) bearing dual functional groups (amino and carboxyl groups) was prepared through etherification of starch with 2-chloro-4,6-diglycino-[1,3,5]-triazine. PRAS exhibits a reversible pH-response property in aqueous solution. The attractive property of PRAS is that it could be used as an effective flocculant for heavy metal-ion (e.g. Cu(ii) and Zn(ii)) removal from wastewater by changing pH. The transition of hydrophobicity–hydrophilicity would produce shrinkage of the polymer matrix, facilitating the release of heavy-metal ions from the saturated flocculant. As an ideal flocculant PRAS displayed outstanding stability and reproducibility, whose remove rate for Cu(ii) and Zn(ii) remained at 93% and 91% after three flocculation/regeneration cycles.

A pH-responsive starch-based flocculants containing both cationic and anionic functional groups has been developed. The saturated flocculant can be facilely regenerated and separated from the solution by applying an external pH stimulus.     

Friedel–Crafts alkylation reaction with fluorinated alcohols as hydrogen-bond donors and solvents

An effective and clean FC alkylation of indoles and electron-rich arenes with β-nitroalkenes in HFIP was reported. The desired products are formed rapidly in excellent yields under mild conditions without the need for any additional catalysts or reagents. Further, this methodology can be applied to one-pot synthesis of biologically active tryptamine derivatives.

Friedel–Craft alkylation of heterocycle derivatives with β-nitroalkenes was performed in HFIP. The one-pot synthesis of tryptamines could be applied.

Friedel–Crafts (FC) alkylation is one of the most important methods for C–C bond formation in organic chemistry.¹ The FC alkylation between indoles and β-nitroalkenes is of particular interest due to the versatility by which the nitro group can be transformed into other functional groups.² Numerous researchers have developed new conditions and effective catalysts in the past decades.³ Among them, H-bond donor (HBD) catalysts have become a focus of an increasing amount of research.⁴ Significant advances have been made in the area of homogeneous HBD catalysts by using urea or thiourea,^4a 2-aminopyridinium ions^4b boronate ureas,^4e and silanediol.^4d,4f However, the strong hydrogen-bonding nature of the HBD catalysts drives a detrimental self-association or aggregation, thus reducing or enhancing catalytic activity.⁵Trifluoroethanol (TFE) and hexafluoro-2-propanol (HFIP), exhibit unique features in organic synthesis. Indeed, the presence of the strong electron-withdrawing trifluoromethyl group influences several key parameters: ionizing power, Brønsted acidity, and hydrogen-bond donation HBD (or H-bond acidity) are increased, whereas nucleophilicity and hydrogen-bond acceptance HBA (or H-bond basicity) are significantly depleted.⁶ Furthermore, the HBD was strongly connected to the conformation along the C–O bond. Moreover, it was suggested that aggregation of HFIP as dimers or trimers could also enhance this property.⁷ Few FC alkylations have been studied solely in HFIP in last years. In 2010, Qu and co-workers described the intra- and intermolecular FC alkylations of electron-rich arenes with epoxides promoted by fluorinated alcohols.⁸ Recently, FC alkylations of arenes with benzyl halides in HFIP have been developed by Paquin et al.⁹ and Khaled et al. (Scheme 1a). More recently, HFIP promoting intramolecular and intermolecular Friedel–Crafts acylation have been reported by Jeffrey Aube and co-workers (Scheme 1b).¹⁰Open in a separate windowScheme 1Examples of FC alkylation promoted by HFIP.In our interest for the use of fluorous medium in organic synthesis,¹¹ and in the pursuit of more efficient catalytic systems, we found that fluorinated alcohols promoted the FC alkylation between indoles and β-nitroalkenes.The FC alkylation between indole (1a) and β-nitroalkene (2a) was used as model system to optimize the reaction conditions (6b With other fluorinated alcohols, perfluoro-2-methylpropan-2-ol (PFTB) was more efficient than the trifluoroethanol (TFE) (entries 10 and 11). In contrast, in EtOH, only traces of product were observed (entry 12). When performed the reaction in the dark had no effect on the yield (entry 13), which suggested HFIP promoted FC alkylation is not a photochemical pathway.¹²Optimization of conditions for the FC alkylation^a

Rapid sequence airway vs rapid sequence intubation in a simulated trauma airway by flight crew

Background

Rapid sequence airway (RSA) utilizes rapid sequence intubation (RSI) pharmacology followed by the placement of an extraglottic airway without direct laryngoscopy.

Study objective

To evaluate the difference in time to airway placement and lowest oxygen saturations in a simulated trauma patient using RSI or RSA with a Laryngeal Mask Airway—Supreme (LMAS).

Methods

This randomized, prospective, non-blinded, IRB-approved observational study used a SimMan^® human simulator in an ambulance. FC were randomly assigned to initially manage the patient with RSI or RSA. They then completed the same scenario with the other modality to serve as their own control. Trained assistants performed directed tasks. SimMan^® had an initial grade III view and desaturated along a standardized curve until intubation, LMAS, or bag-valve-mask ventilation (BVMV) was initiated. When BVMV was used, oxygen saturation increased along a standardized curve. The simulator's airway converted to a grade II view after the first attempt if difficult airway maneuvers were applied. Time, oxygen saturation, number of attempts and back-up airway placement were recorded.

Results

Nineteen FC completed both paired modalities. Paired T-test was used for statistical analysis. Average time to secure the airway was 145 s shorter in the RSA group (95% CI: 100.4-189.7). Lowest oxygen saturation was 4.8% higher (95% CI: 2.8-6.8) in the RSA group. During RSI, FC placed a back-up airway 47% of the time.

Conclusion

In a simulated moderately difficult trauma airway managed by FC, RSA results in a significantly shorter time to secure the airway and less hypoxemia compared to RSI.     

A divergent approach for the synthesis of (hydroxymethyl)furfural (HMF) from spent aromatic biomass-derived (chloromethyl)furfural (CMF) as a renewable feedstock

Extraction of commercial essential oil from several aromatic species belonging to the genus Cymbopogon results in the accumulation of huge spent aromatic waste which does not have high value application; instead, the majority is burned or disposed of to vacate fields. Open burning of spent aromatic biomass causes deterioration of the surrounding air quality. Therefore, a new protocol has been developed for chemical processing of spent biomass to obtain 5-(chloromethyl)furfural (CMF) with high selectivity (∼80%) and yields (∼26 wt% or ∼76 mol% with respect to pre-treated biomass) via refluxing in aqueous HCl in the presence of NaCl as a cheap catalyst. No black tar formation and gasification were observed in the processing of the spent aromatic biomass. Spent aromatic waste-derived CMF was further converted to 5-(hydroxymethyl)furfural (HMF) in good yields by a novel one pot method using iodosylbenzene (PhIO) as a reagent under mild reaction conditions.

Extraction of commercial essential oil from aromatic crops results in the accumulation of huge spent aromatic waste which can be used for production of platform chemicals such as xylose, CMF and HMF.     

Treatment of mild non-chemotherapy-induced iron deficiency anemia in cancer patients: Comparison between oral ferrous bisglycinate chelate and ferrous sulfate

In cancer patients mild-moderate non-chemotherapy-induced iron deficiency anemia (IDA) is usually treated with oral iron salts, mostly ferrous sulfate. In this study, we compare efficacy and toxicity of oral ferrous bisglycinate chelate and ferrous sulfate in cancer patients with mild IDA. Twenty-four patients operated on for solid tumors (10 breast, 12 colorectal, 2 gastric), aged 61 ± 10 years (range 45–75), with non-chemotherapy-induced hemoglobin (Hb) values between 10 and 12 g/dL and ferritin lower than 30 ng/mL were randomized to receive oral ferrous bisglycinate chelate, 28 mg per day for 20 days, and then 14 mg per day for 40 days (12 patients) (A group) or oral ferrous sulphate, 105 mg per day for 60 days (12 patients) (B group). Values of hemoglobin and ferritin obtained at diagnosis, 1 and 2 months from the beginning of treatment were compared. Adverse events (AEs) related to the two treatments were recorded. In the 12 patients treated with ferrous bisglycinate chelate, basal hemoglobin and ferritin values (mean ± SD) were 11.6 ± 0.8 g/dL and 16.1 ± 8.0 ng/mL. After 2 months of treatment, they were 13.0 ± 1.4 g/dL and 33.8 ± 22.0 ng/mL, respectively (P = 0.0003 and P = 0.020). In the group treated with ferrous sulphate, hemoglobin and ferritin mean values were 11.3 ± 0.6 g/dL and 19.0 ± 6.4 ng/mL basally, and 12.7 ± 0.70 g/dL and 40.8 ± 28.1 ng/mL (P < 0.0001 and P = 0.017) after 2 months of treatment. AEs occurred in six cases. In all these six cases, two (17%) treated with ferrous bisglycinate chelate and four (33%) with ferrous sulphate, toxicity was grade 1. In conclusion, these data suggest that ferrous bisglycinate chelate has similar efficacy and likely lower GI toxicity than ferrous sulphate given at the conventional dose of 105 mg per day for the same time.     

The Effects of Water Volume and Bacterial Concentration on the Water Filtration Assay Used in Zebrafish Health Surveillance

The number of zebrafish in biomedical research has increased exponentially over the past decades, leading to pressure on the laboratory animal community to develop and refine techniques to monitor zebrafish health so that suitable stocks can be maintained for research. The water filtration assay is a promising technique in which water from a zebrafish system is filtered, and the filter analyzed by PCR. In the present report, we studied how the volume of water tested and the concentration of bacterial pathogens affected test results. To do so, we used stock solutions of 3 zebrafish pathogens: Edwardsiella ictaluri, Aeromonas hydrophila, and Mycobacterium marinum. We used these stocks to create solutions with known concentrations of each pathogen, ranging between 10² and 10⁷ Colony Forming Units (CFU) per ml. One, 2, and 3 L of each solution was filtered using positive pressure, and the filters were submitted to a commercial lab for PCR testing. Results were fit with a logistic regression model, and the probability of obtaining a positive result were calculated. Test sensitivity varied by organism, but in general, test results were positively correlated with the volume of the water filtered and with the concentration of bacteria in solution. We conclude that a positive result can be expected for E. ictaluri at 10⁵ CFU per mL, A. hydrophila at 10⁶ CFU per ml, and M. marinum at 10⁶ CFU per mL, when 3 L of solution are filtered.

A major goal of laboratory animal medicine is to safeguard the health of research colonies and research staff. Ideally, every animal used in research would be free of all organisms with the potential to confound research by inducing physiologic changes or causing zoonotic disease. To achieve this goal, the health status of animals should be defined and reported. As the use of zebrafish (Danio rerio) in research has grown, the environmental testing and technology necessary to maintain laboratory animals free from pathogenic microbial contamination has also expanded.^{1,3,4,9,12-14} A novel method of testing a zebrafish colony for the presence of certain pathogens used a PCR filter assay in which water was filtered from a zebrafish system and the filters then submitted for PCR analysis.^4,19 The study demonstrated that various microbes in zebrafish system water could be identified using this method.The present report builds on the previous study by conducting experiments designed to show whether the water filtration assay was affected by the volume of water filtered and the concentration of bacteria (Edwardsiella ictaluri, Aeromonas hydrophila, or Mycobacterium marinum) in the water. We selected these 3 organisms based on several factors: 1) each can cause disease in zebrafish,^5-8,17,21 2) A. hydrophila and M. marinum have zoonotic potential,^5,7,8 3) the necessary cultures and supplies are commercially available, and 4) expertise and equipment is available to culture the organisms. Finally, we wanted to determine if we could detect A. hydrophila by PCR analysis due to an outbreak of this organism in one of our colonies that routinely tested negative by other methods.     

Poster 21: Does severe left ventricular dysfunction impact the result of cardiac rehabilitation?

Objective: To evaluate the effect of severe left ventricular dysfunction on the improvement of functional capacity (FC) in cardiac patients undergoing phase 2 of a cardiac rehabilitation program (CRP). Design: Retrospective cohort study. Setting: Hospital-based CRP. Participants: 199 male cardiac patients. Group 1 (n=169) had a left ventricular ejection fraction (LVEF) >30% (age, 66.2±9.8y); group 2 (n=30) had a LVEF ≤30% (age, 69.0±8.1y). Intervention: 10 weeks, thrice weekly, of phase 2 CRP, consisting of 60 minutes of supervised exercise to reach the target heart rate determined by the Karvonen method. Main Outcome Measures: We measured FC before and after completion of the CRP and the improvement expressed in percents of FC before the CRP. The FC results were compared using the Student t test. Results: FC in both patient groups improved after the CRP. In group 1 patients, FC increased from 5.6±2.3 metabolic equivalents (METS) before the CRP to 7.5±2.6 METS after the CRP (P<.01). In group 2 patients, FC increased from 4.7±2.1 METS before the CRP to 6.2±2.2 METS after the CRP (P<.01). Before the CRP, group 2 patients had significantly lower FC compared with group 1 patients (P<.05). Similarly, after the CRP, the FC of group 2 patients remained lower than FC of group 1 patients (P<.05). However, the percentage of improvement for group 1 patients (40.6%±34.3%) did not differ significantly from the percentage of improvement for group 2 patients (39.9%±34.5%). Conclusions: The CRP improved FC of all cardiac patients, including those with severe left ventricular dysfunction. Patients with severe left ventricular dysfunction have lower FC before and after the CRP. However, the FC of these patients improved to the same degree as the patients with better left ventricular function. These findings are important in designing strategies for the CRP in patients with severely impaired LVEF.     

Silica-immobilized ionic liquid Brønsted acids as highly effective heterogeneous catalysts for the isomerization of n-heptane and n-octane

Metal-free imidazolium-based ionic liquid (IL) Brønsted acids 1-methyl imidazolium hydrogen sulphate [HMIM]HSO₄ and 1-methyl benzimidazolium hydrogen sulphate [HMBIM]HSO₄ were synthesized. Their physicochemical properties were investigated using spectroscopic and thermal techniques, including UV-Vis, FT-IR, ¹H NMR, ¹³C-NMR, mass spectrometry, and TGA. The ILs were immobilized on mesoporous silica gel and characterized by FT-IR spectroscopy, scanning electron microscopy, Brunauer–Emmett–Teller analysis, ammonia temperature-programmed desorption, and thermogravimetric analysis. [HMIM]HSO₄@silica and [HMBIM]HSO₄@silica have been successfully applied as promising replacements for conventional catalysts for alkane isomerization reactions at room temperature. Isomerization of n-heptane and n-octane was achieved with both catalysts. In addition to promoting the isomerization of n-heptane and n-octane (a quintessential reaction for petroleum refineries), these immobilized catalysts are non-hazardous and save energy.

Metal-free imidazolium-based ionic liquid (IL) Brønsted acids 1-methyl imidazolium hydrogen sulphate [HMIM]HSO₄ and 1-methyl benzimidazolium hydrogen sulphate [HMBIM]HSO₄ were synthesized.     

Surface blocking of azolla modified copper electrode for trace determination of phthalic acid esters as the molecular barricades by differential pulse voltammetry: response surface modelling optimized biosensor

In this work, a sensitive and efficient voltammetric biosensor was introduced for differential pulse voltammetric (DPV) determination of some phthalic acid esters (PAEs) including dibutyl phthalate (DBP), dimethyl phthalate (DMP), di(2-ethylhexyl)phthalate (DEHP) and dicyclohexyl phthalate (DCHP) in aqueous solutions. Briefly, the surface of a copper electrode was modified by azolla paste prepared using azolla powder and electroencephalography gel (EEG). The modified surface was characterized by electrochemical impedance spectroscopy (EIS), field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), Brunauer–Emmett–Teller (BET) analysis and energy dispersive X-ray (EDX) methods. Determination of PAEs was conducted based on their blocking effect on the electrode surface for ferrous ion oxidation. The central composite design (CCD) was conducted to optimize the effects of four experimental parameters including the concentration of Fe²⁺ ions (C_Fe²⁺) and supporting electrolyte (C_{sup. elec}), solution pH and modifier/gel mass ratio on the decrease in the anodic peak current of ferrous ions as the response. Predicted optimal conditions (C_Fe²⁺= 319 μM, C_{sup. elec}= 0.125 M, pH = 7.52 and modifier/gel mass ratio = 0.19) were validated by experimental checking which resulted in an error of 1.453%. At the optimum conditions, linear relationships were found between the DPV responses and PAEs concentrations and the limit of detection (LOD) and limit of quantification (LOQ) values were in the ranges of 0.2–0.4 μg L⁻¹ and 0.5–1.0 μg L⁻¹, respectively. Good recovery percentages ranging from 97.3 to 100.3% with RSD < 3.2% suggested the proposed method for efficient, accurate and quick determination of PAEs in real water samples.

In this work, a sensitive and efficient voltammetric biosensor was introduced for differential pulse voltammetric (DPV) determination of dibutyl phthalate, dimethyl phthalate, di(2-ethylhexyl)phthalate and dicyclohexyl phthalate in aqueous solutions.     

pH-responsive chitosan-based flocculant for precise dye flocculation control and the recycling of textile dyeing effluents

In this work, we introduce a simple and effective method for the controlled release of dye from dye saturation flocs by a well-designed pH responsive chitosan-based flocculant. The dye flocculation capacities could be precisely controlled from 0.5 to 2 g g⁻¹ by simply adjusting the pH of the desorption solution. A series of flocs with different dye flocculation capacities was prepared and used as nitrogen-rich precursors to prepare nitrogen-doped carbon materials through one-step carbonization. The results demonstrate that the specific surface areas, pore structures and supercapacitance performance of the resulting N-doped carbon materials could be readily controlled by varying the dye flocculation capacity. By using a dye sludge floc with an appropriate dye flocculation capacity (1.5 g g⁻¹) as a precursor, the resulting N-doped material exhibited a high specific capacity and good cycling performance for a supercapacitor electrode. The unique pH-responsive properties of the chitosan-based flocculant facilitated easy tuning of the surface cationic degree and deprotonation behavior by varying pH. This work presents a new concept for balancing between environmental capacity and energy capacity using a smart pH-responsive carrier system based on modified chitosan, which is highly promising for the recycling of industrial wastewater to produce energy materials.

Balance between environmental capacity and energy capacity using a pH-responsive chitosan-based flocculant.     

Noninvasive assessment of hemodynamic response to a fluid challenge using femoral Doppler in critically ill ventilated patients

Purpose

The purpose of the study is to determine if femoral artery blood flow Doppler parameters can assess cardiac response to a fluid challenge (FC).

Materials and Methods

We prospectively recorded in 52 critically ill ventilated patients' velocity time integral variation (%VTIf) and maximal systolic velocity variation (%Vfmax) derived from femoral Doppler analysis and aortic velocity time integral variation registered on transthoracic echocardiography before and after an FC of 500-mL saline.

Results

According to Pearson coefficient, %Vfmax and %VTIf were found to be positively correlated with aortic velocity time integral variation (r² = 0.46 and 0.51, respectively; P < .0001) and were significantly different between responder patients and nonresponders (11% ± 3.4% vs 5.9% ± 4.3% and 14.9% ± 4.2% vs 5.5% ± 5.5%, respectively; P < .0001). Increase of %VTIf 10% or higher and %Vfmax 7% or higher after an FC showed a sensitivity of 80% and 84%, a specificity of 85% and 73%, and an area under the curve of 0.905 and 0.851, respectively, for discriminating responder and nonresponder patients.

Conclusion

Variation of femoral Doppler parameters before and after FC mirrors cardiac response to fluid loading. This tool could be considered as an alternative to transthoracic echocardiography in case of poor thoracic insonation.     

Fluorometric trace methanol detection in ethanol and isopropanol in a water medium for application in alcoholic beverages and hand sanitizers

Detection of methanol (MeOH) in an ethanol (EtOH)/isopropanol (ⁱPrOH) medium containing water is crucial to recognize MeOH poisoning in alcoholic beverages and hand sanitizers. Although chemical sensing methods are very sensitive and easy to perform, the chemical similarities between the alcohols make MeOH detection very challenging particularly in the presence of water. Herein, the fluorometric detection of a trace amount of MeOH in EtOH/ⁱPrOH in the presence of water using alcohol coordinated Al(iii)-complexes of an aldehydic phenol ligand containing a dangling pyrazole unit is described. The presence of MeOH in the EtOH/ⁱPrOH causes a change of the complex geometry from tetrahedral (Td) to octahedral (Oh) due to the replacement of the coordinated EtOH/ⁱPrOH by MeOH molecules. The Td-complex exhibited fluorescence but the Oh-species did not, because of the intramolecular photo-induced electron transfer (PET). By interacting the Oh species with water, its one MeOH coordination is replaced by a water molecule followed by the proton transfer from the water to pyrazole-N which generates strong fluorescence by inhibiting the PET. In contrast, the water interaction dissociates the Td-complex to exhibit fluorescence quenching. The water induced reversal of the fluorescence response from the decrease to increase between the absence and presence of MeOH is utilized to detect MeOH in an EtOH/ⁱPrOH medium containing water with a sensitivity of ∼0.03–0.06% (v/v). The presence of water effected the MeOH detection and allows the estimation of the MeOH contamination in alcoholic beverages and hand sanitizers containing large amounts of water.

Detection of MeOH in EtOH/ⁱPrOH and commercial samples based on water induced reversal of fluorescence response from the decrease to increase between the absence and presence of MeOH.