SUSPECTED CARBARYL TOXICITY IN A CAPTIVE COLONY OF STRAW-COLORED FRUIT BATS ( EIDOLON HELVUM).

Carbaryl was the first carbamate insecticide produced and remains the most widely used due to its perceived low level of toxicity in nontarget species. This report describes the management and evaluation of a group of straw-colored fruit bats, Eidolon helvum, that were exposed to carbaryl. Cholinesterase activity of blood, retina, and brain was evaluated to further investigate whether carbaryl was the causative agent. Decreased whole blood and retinal cholinesterase activity coupled with the response to atropine suggests that the cause of the clinical signs in this bat colony was due to carbaryl exposure. Whole blood and retinal tissue may be the best samples for confirming carbamate exposure in this species.

ESTABLISHMENT OF REFERENCE INTERVALS FOR HEMATOLOGY AND BIOCHEMISTRY ANALYTES IN A CAPTIVE COLONY OF STRAW-COLORED FRUIT BATS (EIDOLON HELVUM).

Straw-colored fruit bats (Eidolon helvum) are an ecologically important species of megachiropteran bat that are common both in captivity and in the wild. This study attempts to determine reference intervals from a group of 45 captive individuals for a variety of hematologic and biochemistry parameters. Most of the values are similar to values previously seen in other pteropodid bat species. Differences included a lower than expected reference interval for the total white blood cell count, a wider reference interval for glucose, and a higher reference interval for 25-hydroxyvitamin D. Establishing reference intervals for hematologic and biochemistry analytes for bats that were maintained on the same diet and under the same husbandry parameters for several years will help in the future assessment of other bats maintained in a similar manner.

Debranching of soluble wheat arabinoxylan dramatically enhances recalcitrant binding to cellulose.

The presence of xylan is a detriment to the enzymatic saccharification of cellulose in lignocelluloses. The inhibition of the processive cellobiohydrolase Cel7A by soluble wheat arabinoxylan is shown here to increase by 50% following enzymatic treatment with a commercially-purified α-L-arabinofuranosidase. The enhanced inhibitory effect was shown by T2 relaxation time measurements via low field NMR to coincide with an increasing degree of constraint put on the water in xylan solutions. Furthermore, quartz crystal micro-balance with dissipation experiments showed that α-L-arabinofuranosidase treatment considerably increased the rate and rigidity of arabinoxylan mass association with cellulose. These data also suggest significant xylan-xylan adlayer formation occurs following initial binding of debranched arabinoxylan. From this, we speculate the inhibitory effects of xylan to cellulases may result from reduced enzymatic access via the dense association of xylan with cellulose.

MANAGEMENT OF OMPHALOPHLEBITIS AND UMBILICAL HERNIA IN THREE NEONATAL GIRAFFE (GIRAFFA CAMELOPARDALIS).

Umbilical disorders, including omphalophlebitis, omphaloarteritis, external umbilical abscesses, urachal abscesses, patent urachus, and umbilical hernias, represent a significant challenge to the health and well-being of a neonate. The three neonatal giraffe (Giraffa camelopardalis) in this report were evaluated for umbilical swellings. Two developed omphalophlebitis, and one had an uncomplicated umbilical hernia. Omphalophlebitis is an inflammation and/or infection of the umbilical vein. Giraffe calves with a failure of passive transfer may be predisposed and should be thoroughly evaluated for the condition. Umbilical hernias result from a failure of the umbilical ring to close after parturition or from malformation of the umbilical ring during embryogenesis. These problems were surgically corrected for all three individuals, although one died due to postsurgical complications. The risks involved include anesthetic complications, surgical dehiscence, and maternal rejection. Early detection and surgical intervention are recommended for the correction of omphalophlebitis and umbilical hernias in neonatal giraffe.

Impact of lignins isolated from pretreated lignocelluloses on enzymatic cellulose saccharification.

Lignins were enzymatically isolated from corn stover and wheat straw samples and subjected to hydrothermal or wet oxidation pretreatments for enzyme adsorption experimentations. Lignin contents of the isolates ranged from 26 to 71 % (w/w); cellulose ranged from 3 to 22 % (w/w); xylan from 0.7 to 6 % (w/w) and ash was from 5.8 to 30 % (w/w). ATR-IR analyses indicated significant and similar levels of calcium in all lignin isolates. Commercial cellulase adsorption studies showed that the presence of these lignins had no significant impact on the total amount of adsorbed enzyme in cellulose and cellulose-lignin systems. Consequently, the presence of the lignins had minimal effect, if any, on enzymatic cellulose conversion. Furthermore, this result, coupled with significant calcium levels in the isolated lignins, supports previous work suggesting lignin-calcium complexes reduce enzyme-lignin interactions.

Hydration and saccharification of cellulose Iß, II and III(I) at increasing dry solids loadings.

Crystalline cellulose Iß (Avicel) was chemically transformed into cellulose II and III(I) producing allomorphs with similar crystallinity indices (ATR-IR and XRD derived). Saccharifications by commercial cellulases at arrayed solids loadings showed cellulose III(I) was more readily hydrolysable and less susceptible to increased dry solids levels than cellulose Iß and II. Analysis by dynamic vapor sorption revealed cellulose II has a distinctively higher absorptive capacity than cellulose I and III(I). When equally hydrated (g water/g cellulose), low-field nuclear magnetic resonance (LF-NMR) relaxometry showed that cellulose II, on average, most constrained water while cellulase III(I) left the most free water. LF-NMR spin-spin relaxation time distribution profiles representing distinct water pools suggest cellulose III(I) had the most restricted pool and changes in water distribution during enzymatic saccharification were most dramatic with respect to cellulose III(I) compared to celluloses Iß and II.

Detection of cracks in concrete using near-IR fluorescence imaging.

Structural health monitoring of civil infrastructure is a crucial component of assuring the serviceability and integrity of the built environment. A primary material used in the construction of civil infrastructure is concrete, a material that is susceptible to cracking due to a variety of causes, such as shrinkage, creep, overloading, and temperature change. Cracking reduces the durability of concrete structures, as it allows deleterious environmental agents to penetrate the surface, causing such damage as corrosion of steel reinforcement and delamination of the concrete itself. Conventional crack detection techniques are limited in scope due to issues relating to pre-planning, accessibility, and the need for close proximity to the test surface. Contactless optical image monitoring techniques offer the opportunity to overcome these limitations and have the potential to detect cracks at a distance. Concrete has been reported to have a near-infrared (Near-IR) fluorescence line at a wavelength of 1140 nm when excited with red light. This work investigates the use of fluorescence imaging for the detection of cracks in cementitious surfaces using shallow angle incidence excitation red light. Light oriented at a shallow angle does not excite interior surfaces of cracks, which appear as darker features in images of fluorescing concrete. Artificial cracks with widths of 0.2-1.5 mm were readily imaged using a near-IR camera at distances of 0.5 and 1.3 m. An additional concrete sample with a 0.08 mm wide crack was produced using a flexure apparatus and was also imaged. It is worth noting that the 0.08 mm crack was detected despite its width being below the 0.1 mm pixel resolution of the camera, with the aid of digital image enhancement algorithms.

Precipitation of Trichoderma reesei commercial cellulase preparations under standard enzymatic hydrolysis conditions for lignocelluloses.

Comparative studies between commercial Trichoderma reesei cellulase preparations show that, depending on the preparation and loading, total protein precipitation can be as high as 30 % under standard hydrolysis conditions used for lignocellulosic materials. ATR-IR and SDS-PAGE data verify precipitates are protein-based and contain key cell wall hydrolyzing enzymes. Precipitation increased considerably with incubation temperature; roughly 50-150 % increase from 40 to 50 °C and 800 % greater at 60 °C. All of the reported protein losses translated into significant, and often drastic, losses in activity on related 4-nitrophenyl substrates. In addition, supplementation with the non-ionic surfactant PEG 6,000 decreased precipitation up to 80 % in 24 h precipitation levels. Protein precipitation is potentially substantial during enzymatic hydrolysis of lignocelluloses and should be accounted for during lignocellulose conversion process design, particularly when enzyme recycling is considered.

Antisense down-regulation of 4CL expression alters lignification, tree growth, and saccharification potential of field-grown poplar.

Transgenic down-regulation of the Pt4CL1 gene family encoding 4-coumarate:coenzyme A ligase (4CL) has been reported as a means for reducing lignin content in cell walls and increasing overall growth rates, thereby improving feedstock quality for paper and bioethanol production. Using hybrid poplar (Populus tremula × Populus alba), we applied this strategy and examined field-grown transformants for both effects on wood biochemistry and tree productivity. The reductions in lignin contents obtained correlated well with 4CL RNA expression, with a sharp decrease in lignin amount being observed for RNA expression below approximately 50% of the nontransgenic control. Relatively small lignin reductions of approximately 10% were associated with reduced productivity, decreased wood syringyl/guaiacyl lignin monomer ratios, and a small increase in the level of incorporation of H-monomers (p-hydroxyphenyl) into cell walls. Transgenic events with less than approximately 50% 4CL RNA expression were characterized by patches of reddish-brown discolored wood that had approximately twice the extractive content of controls (largely complex polyphenolics). There was no evidence that substantially reduced lignin contents increased growth rates or saccharification potential. Our results suggest that the capacity for lignin reduction is limited; below a threshold, large changes in wood chemistry and plant metabolism were observed that adversely affected productivity and potential ethanol yield. They also underline the importance of field studies to obtain physiologically meaningful results and to support technology development with transgenic trees.

High throughput determination of glucan and xylan fractions in lignocelluloses.

The analysis of structural glucan and xylan in lignocellulose was scaled down from original two-stage sulfuric acid hydrolysis methods (Moore WE and Johnson DB 1967 Procedures for the chemical analysis of wood and wood products. U.S. Forest Products Laboratory, U.S. Department of Agriculture., Madison, WI) and integrated into a recently-developed, high throughput pretreatment and enzymatic saccharification system. Novel 96×1.8 ml-well Hastelloy reactor plates (128×86×51 mm) based on previously described 96-well pretreatment reactor plates were paired with custom aluminum filler plates (128×86×18 mm) for use in Symyx Powdernium solids dispensing systems. The incorporation of glucose oxidase and xylose dehydrogenase linked assays to speed post-hydrolysis sugar analysis dramatically reduced the time for analysis of large lignocellulosic sample sets. The current system permits the determination of the glucan and xylan content of 96 replicates (per reactor plate) in under 6 h and parallel plate processing increases the analysis throughput substantially.

Detecting cellulase penetration into corn stover cell walls by immuno-electron microscopy.

In general, pretreatments are designed to enhance the accessibility of cellulose to enzymes, allowing for more efficient conversion. In this study, we have detected the penetration of major cellulases present in a commercial enzyme preparation (Spezyme CP) into corn stem cell walls following mild-, moderate- and high-severity dilute sulfuric acid pretreatments. The Trichoderma reesei enzymes, Cel7A (CBH I) and Cel7B (EG I), as well as the cell wall matrix components xylan and lignin were visualized within digested corn stover cell walls by immuno transmission electron microscopy (TEM) using enzyme- and polymer-specific antibodies. Low severity dilute-acid pretreatment (20 min at 100 degrees C) enabled <1% of the thickness of secondary cell walls to be penetrated by enzyme, moderate severity pretreatment at (20 min at 120 degrees C) allowed the enzymes to penetrate approximately 20% of the cell wall, and the high severity (20 min pretreatment at 150 degrees C) allowed 100% penetration of even the thickest cell walls. These data allow direct visualization of the dramatic effect dilute-acid pretreatment has on altering the condensed ultrastructure of biomass cell walls. Loosening of plant cell wall structure due to pretreatment and the subsequently improved access by cellulases has been hypothesized by the biomass conversion community for over two decades, and for the first time, this study provides direct visual evidence to verify this hypothesis. Further, the high-resolution enzyme penetration studies presented here provide insight into the mechanisms of cell wall deconstruction by cellulolytic enzymes.

Synergistic enhancement of cellobiohydrolase performance on pretreated corn stover by addition of xylanase and esterase activities.

Significant increases in the depolymerization of corn stover cellulose by cellobiohydrolase I (Cel7A) from Trichoderma reesei were observed using small quantities of non-cellulolytic cell wall-degrading enzymes. Purified endoxylanase (XynA), ferulic acid esterase (FaeA), and acetyl xylan esterase (Axe1) all enhanced Cel7A performance on corn stover subjected to hot water pretreatment. In all cases, the addition of these activities improved the effectiveness of the enzymatic hydrolysis in terms of the quantity of cellulose converted per milligram of total protein. Improvement in cellobiose release by the addition of the non-cellulolytic enzymes ranged from a 13-84% increase over Cel7A alone. The most effective combinations included the addition of both XynA and Axe1, which synergistically enhance xylan conversions resulting in additional synergistic improvements in glucan conversion. Additionally, we note a direct relationship between enzymatic xylan removal in the presence of XynA and the enhancement of cellulose hydrolysis by Cel7A.

Anthocyanin stabilization by chitosan-chondroitin sulfate polyelectrolyte complexation integrating catechin co-pigmentation.

The thermochemical instability of anthocyanins (ATC) presents a challenge to their utilization as natural colorants in many food systems. This is addressed herein with the development of polysaccharide based carriers formed by combined encapsulation and copigmentation approaches which utilize polyelectrolyte complexation between chitosan and chondroitin sulfate (CS). At pH 3, a 1.5mg/mL and 1:1wt ratio mix of both polysaccharides produced hydrophilic and positively charged polyelectrolyte complexes (PECs) with which a maximum ATC encapsulation efficiency of 88% could be achieved using a 1:6 elderberry extract as the ATC source. ATC coupled with EGCG co-pigmentation achieved the highest encapsulation efficiencies. Storage studies showed the combination of polysaccharide encapsulation and EGCG copigmentation improved ATC stability against elevated temperature and ascorbic acid. Copigmented PECs were shown to retain ATC color at a rate more than 3-fold greater than of non-encapsulated ATC, and, furthermore, were shown to improve and preserve ATC anti-oxidant activity and stability during storage.

Formation of shelf stable Pickering high internal phase emulsions (HIPE) through the inclusion of whey protein microgels.

High internal phase emulsions (HIPE) prepared using whey protein microgels (WPMs) as a surfactant were demonstrated to have substantially higher stability than HIPEs prepared using similar loadings of non-gelled whey protein isolate (WPI) or Tween 20. Microgel colloids were prepared from WPI solutions by heat treatment at 85 °C in a narrow pH range (5.8-6.0) to particle sizes of approximately 90, 160 and 350 nm in diameter. ζ-potentials of the WPM increased in negativity with decreasing particle size from -7.4 ± 2.5 down to -21.1 ± 0.9 at 90 nm. All WPMs conferred high stability to corn oil based HIPE when used as an emulsifier. Light microscopy and cryo-scanning electron microscopy showed that both increasing WPM concentration and decreasing WPM particle size produced increasingly smaller and more hexagonally shaped corn oil emulsion droplets; WPI and Tween 20 based HIPE droplets were generally smaller and spherical in shape. The HIPE (75% w/w corn oil) produced with 1% (w/w) WPM as an emulsifier showed stability through 6 months storage at 4 °C at all WPM sizes tested, while the HIPE prepared with 1% (w/w) WPI or Tween 20 exhibited significant creaming. WPM and WPI based HIPE both showed thermal stability at 70 °C and 95 °C while the heating of Tween 20 based HIPE resulted in droplet coalescence and oil-phase separation. HIPE production with WPMs significantly improved the viscoelastic properties of the HIPE, imparting drastic increases in yield stress, critical stress, complex modulus and elastic modulus over HIPE prepared with WPI or Tween 20. The more rigid rheology of the WPM HIPE indicated by these data is likely the primary mechanism driving the improved stability of these emulsions.

Catechin modulates the copigmentation and encapsulation of anthocyanins in polyelectrolyte complexes (PECs) for natural colorant stabilization.

Our objective was to develop a robust system for anthocyanin-based color intensification, with high-encapsulation capacity and improved stability of the encapsulated natural colorant. Catechin was used to modulate the copigmentation and encapsulation of anthocyanins in counter-ionic polyelectrolyte complexes (PECs) composed of chondroitin sulfate and chitosan. Results showed that catechin copigmentation significantly intensified red color of formulations both with and without encapsulation in PECs and improved the anthocyanin encapsulation efficiency by forming additional dense network through hydrogen bonding. A series of stability assays revealed that anthocyanin stabilizing effect of system depended on the formulated pH and adding order of catechin. The strongest retaining capacity of anthocyanin was observed when catechin was copigmented with anthocyanin directly in PECs at pH 3.3, while the coated layer of catechin covered on PECs would be more effective at pH 4.0 and 5.0. Furthermore, we demonstrated that this delivery system works for anthocyanins from different sources.

Polyelectrolyte microcapsules built on CaCO3 scaffolds for the integration, encapsulation, and controlled release of copigmented anthocyanins.

The all-polysaccharide based polyelectrolyte microcapsules combining copigmentation for anthocyanin encapsulation and stabilization were fabricated. Copigmented complexes of chondroitin sulfate and anthocyanin were preloaded in CaCO3 scaffold, and then microcapsules were created by coating the sacrificial CaCO3 using layer-by-layer technique. It was observed that the preloading of copigmented complex affected the precipitation reaction of CaCO3 and the subsequent entrapment of anthocyanin. With addition of anthocyanin from 0.125 to 0.75 mg, copigmentation can significantly increase the encapsulation efficiency of anthocyanin in CaCO3, whereas such effect was not obvious at higher loadings. The leakage of anthocyanin during CaCO3 core dissolution and storage was also inhibited by two polysaccharide layers coupled with copigmentation, which may be related to the formation of interconnecting networks. Additionally, a higher anthocyanin antioxidant activity was provided by carbohydrate matrix. These findings may allow for the encapsulation of large amounts of water-soluble components; particularly natural colorant by copigmented complex-polyelectrolyte structures.

Transglutaminase-treated conjugation of sodium caseinate and corn fiber gum hydrolysate: Interfacial and dilatational properties.

This study compliments previous work where peroxidase was successfully used to crosslink corn fiber gum (CFG) with bovine serum albumin and improve CFG's emulsifying properties. Herein, an alternative type of enzyme, transglutaminase, was used to prepare conjugates of CFG and sodium caseinate. Additionally, the CFG was partially hydrolyzed by sulfuric acid and its crosslinking pattern with caseinate was evaluated. The interfacial crosslinking degree between caseinate and CFG increased after hydrolysis according to high performance size exclusion chromatography. The equilibrium interfacial tension of CFG hydrolysate-caseinate conjugate was lower than that of CFG-caseinate conjugate as the rearrangement rate of the CFG hydrolysate-caseinate conjugate was higher. The dilatational modulus of CFG hydrolysate decreased from that of CFG.

Modulation of whey protein-kappa carrageenan hydrogel properties via enzymatic protein modification.

Treatment of whey protein isolate (WPI; 1 to 25% w/w) in heated κ-carrageenan (KC; 2% w/w) slurries with protease and/or transglutaminase modulated the properties of the hydrogels formed after cooling. Observation of peak compression stress and strain at gel rupture showed WPI incorporation at 1, 5 and 10% (w/w) significantly reduced the strength and deformability of 2% (w/w) KC gels. Treatment of WPI solutions in KC slurries with Alcalase 2.4L was shown by both SDS-Page and size exclusion HPLC to reduce protein/peptide molecular weight distributions below 10 kDa, with large portions below 1 kDa. This peptide size reduction within the KC matrix produced more translucent gels with a more organized wall and cell structure as observed by SEM, which resulted in gels with observed rupture stress/strain levels similar to 2% KC alone. Transglutaminase treatment of WPI-KC slurries showed the reverse behavior, reducing gel translucency, strength and deformability. At these loadings, WPI-KC gel strength/deformability appears to relate decreasing peptide size to gel behavior trending towards KC-only gels; suggesting peptide size modulation in protein-carbohydrate complexes will allow significant tailoring of texture for the delivery of protein/peptide rich gelled products.

Corrigendum: Early-Onset Progressive Retinal Atrophy Associated with an IQCB1 Variant in African Black-Footed Cats (Felis nigripes).

This corrects the article DOI: 10.1038/srep43918.

Deposition of lignin droplets produced during dilute acid pretreatment of maize stems retards enzymatic hydrolysis of cellulose.

Electron microscopy of lignocellulosic biomass following high-temperature pretreatment revealed the presence of spherical formations on the surface of the residual biomass. The hypothesis that these droplet formations are composed of lignins and possible lignin carbohydrate complexes is being explored. Experiments were conducted to better understand the formation of these "lignin" droplets and the possible implications they might have on the enzymatic saccharification of pretreated biomass. It was demonstrated that these droplets are produced from corn stover during pretreatment under neutral and acidic pH at and above 130 degrees C, and that they can deposit back onto the surface of residual biomass. The deposition of droplets produced under certain pretreatment conditions (acidic pH; T > 150 degrees C) and captured onto pure cellulose was shown to have a negative effect (5-20%) on the enzymatic saccharification of this substrate. It was noted that droplet density (per unit area) was greater and droplet size more variable under conditions where the greatest impact on enzymatic cellulose conversion was observed. These results indicate that this phenomenon has the potential to adversely affect the efficiency of enzymatic conversion in a lignocellulosic biorefinery.