Longer Digestion Times Research Articles

Circular utilization of discarded oyster farming bamboo scaffolding in pulp and papermaking

Oyster Farming is one of important fisheries and aquaculture industries in Taiwan. Each year, approximately 4000–5000 tons of discarded bamboo scaffolding (BS) used in oyster farming, are generated, so the treatment and utilization of BS should be taken seriously. This study evaluates the suitability of BS for pulp and papermaking by assessing the chemical compositions, microstructural, and fiber morphology. The pulping properties is investigated by soda pulping. The chemical composition of BS shows the potential for application in pulping. The BS microstructure shows that can enhance pulping reactions, while the fiber morphology indicates the possibility of producing high-strength paper. Through the pulping experiment, it demonstrated that BS is suitable for pulping with lower NaOH dosage and longer digestion time. The condition at 170 °C with 14% NaOH dosage for 90 min digestion has the highest yield. After refining the highest pulping yield BS pulp, it can improve the handsheet strength and bulk of the OCC-BS mixed pulp, which can achieve the strength property required for industrial paper. In summary, BS exhibits the potential for pulping application and produces a better paper strength than OCC pulp, exhibiting the feasibility of enhancing the circular utilization value of BS in Taiwan.

Fully Unattended Online Protein Digestion and LC-MS Peptide Mapping.

LC-MS based peptide mapping, i.e., proteolytic digestion followed by LC-MS/MS analysis, is the method of choice for protein primary structural characterization. Manual proteolytic digestion is usually a labor-intensive procedure. In this work, a novel method was developed for fully automated online protein digestion and LC-MS peptide mapping. The method generates LC-MS data from undigested protein samples without user intervention by utilizing the same HPLC system that performs the chromatographic separation with some additional modules. Each sample is rapidly digested immediately prior to its LC-MS analysis, minimizing artifacts that can grow over longer digestion times or digest storage times as in manual or automated offline digestion methods. In this report, we implemented the method on an Agilent 1290 Infinity II LC system equipped with a Multisampler. The system performs a complete digestion workflow including denaturation, disulfide reduction, cysteine alkylation, buffer exchange, and tryptic digestion. We demonstrated that the system is capable of digesting monoclonal antibodies and other proteins with excellent efficiency and is robust and reproducible and produces fewer artifacts than manually prepared digests. In addition, it consumes only a few micrograms of material as most of the digested sample protein is subjected to LC-MS analysis.

Generic detection of organophosphorus nerve agent adducts to butyrylcholinesterase in plasma using liquid chromatography-tandem mass spectrometry combined with an improved procainamide-gel separation and pepsin digestion method

Organophosphorus nerve agent (OPNA) adducts to butyrylcholinesterase (BChE) can be applied to confirm exposure in humans. A sensitive method for generic detection of G- and V-series OPNA adducts to BChE in plasma was developed by combining an improved procainamide-gel separation (PGS) and pepsin digestion protocol with ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Residual matrix interferences from prior PGS purification of OPNA-BChE adducts from plasma were found to be a critical cause of significantly reduced UHPLC-MS/MS detection sensitivity. In our developed on-column PGS approach, the matrix interference was successfully removed by adding an appropriate concentration of NaCl to the washing buffer, and it could capture ≥92.5% of the BChE in plasma. The lower pH value and the longer digestion time in all previous pepsin digestion methods were found to be a key accelerated aging factor of several adducts such as tabun (GA)-, cyclohexylsarin (GF)-, and soman (GD)-BChE nonapeptide adducts, making them difficult to detect. The aging event of several OPNA-BChE nonapeptide adducts was so successfully addressed that the formic acid level in enzymatic buffer and digestion time were lowered to 0.05% (pH 2.67) and 0.5 h, respectively, and the post-digestion reaction was immediately terminated. The improved condition parameters were optimal for pepsin digestion of all types of OPNA-BChE adducts into their individual unaged nonapeptide adducts with the highest yields, expanding the applicability of the method. The method had a nearly one-fold decrease in sample preparation time through the reduction of digestion time and removal of ultrafiltration procedure after digestion. The limit of identification (LOI) were determined respectively as 0.13 ng mL−1, 0.28 ng mL−1, 0.50 ng mL−1, 0.41 ng mL−1 and 0.91 ng mL−1 for VX-, sarin (GB)-, GA-, GF-, and GD-exposed human plasma, being low exposure value compared to previously documented approaches. The approach was utilized to fully characterize the adducted (aged and unaged) BChE levels of five OPNAs in a series of their individual exposed concentration (1.00–400 nM) of plasma sample, and successfully detect OPNA exposure from all unknown plasma samples from OPCW's second and third biomedical proficiency tests. The OPNA-BChE adducts, their aged adducts, and unadducted BChE from OPNA-exposed plasma can simultaneously be measured using the method. The study provides a recommended diagnostic tool for generic verification of any OPNA exposure with high confidence by detecting its corresponding BChE adduct.

Abstract 150: A 515 tumor specimens collection from surgery to benchtop: the WFORCE organoid experience in personalized research

Abstract Introduction: Patient tumor organoids (PTOs) are a new and developing preclinical model in cancer research. However, there is variation in protocols between centers and lack of consistency in important variables, impacting PTO culture success. In this study, we analyzed factors in clinical data and tumor tissue processing to determine optimal conditions for cell isolation and PTO success. Methods: Under IRB approval, tumor tissues from primary and metastatic sites were procured in clinically indicated surgeries then dissociated using a standardized protocol. PTOs were fabricated using collagen-hyaluronic acid ECM and underwent drug screening at 10 days post fabrication, where viability was assessed. Clinical and tissue processing inputs such as type of primary, peritoneal carcinomatosis index (PCI), prior surgery or neoadjuvant chemotherapy, presence of mucin, tissue digestion time, specimen mass, digestion time, and the diameter of isolated cells were analyzed. These factors were assessed by calculating the Pearson Correlation Coefficient (r) and best fit modeling to determine the effect on outputs including tissue viability as measured by % of viable cells using an automated cellular counter, viable cells per mg tissue, and PTO log10 viability by Promega 3D Cell Titer Glo assay. Results: From December 2018-July 2022, 515 tumors from 286 patients with 25 primary types were processed for cellular isolation and PTO fabrication. 407 (79%) provided PTOs for therapeutic testing, with 354 (69%) providing sufficiently viable PTOs for therapeutic interrogation. The most common primary tumor type accrued was appendiceal cancer (n=223, 43%). Tissues processed at 24 hours did not differ in viability outputs when compared to tissues processed immediately (p>0.05). Tissue % viability was better maintained in larger specimens (r=.18, p<0.001), resulting in more viable cells per mg of tissue (r=.12, p=0.009) while it was negatively related to larger cell diameter (r=-.32, p<0.001). The number of viable cells per mg of tumor correlated to higher post-processing cell counts (r=.49, p<0.001) while it was decreased by longer digestion times (r=-.11, p=0.017). Finally, PTO log10 viability, correlated with higher burden of disease as measured by PCI scores (r=.35, p<0.001), larger specimens (r=.19, p<0.001), mucinous tumors (PTO log10 viability 5.46 vs 5.17, p=0.01), and tissues with higher cellular viability (r=.31, p<0.001), while it was negatively impacted by neoadjuvant chemotherapy (5.06 vs 5.41, p<0.001). Conclusions: Higher rates of successful PTO fabrication are obtained in patients with untreated tumors, more aggressive biologic behavior, and increased tumor burden. Creating a unified protocol for tissue processing, agnostic to tumor type, will facilitate incorporation of tissue from the operating room to research protocols with wider translational implications. Citation Format: Steven Donald Forsythe, Richard A. Erali, Preston Laney, William Meeker, Salomat Abdulkhuseynova, Nadeem Wajih, Nicholas Edenhoffer, Cecilia R. Schaaf, Hemamyammal Sivakumar, Aleksander Skardal, Roy E. Strowd, Stephen B. Tatter, Clancy J. Clark, Reese W. Randle, Perry Shen, Edward A. Levine, Ralph B. D'Agostino, Shay Soker, Konstantinos I. Votanopoulos. A 515 tumor specimens collection from surgery to benchtop: the WFORCE organoid experience in personalized research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 150.

Effects of electrokinetic and ultrasonication pre-treatment and two-step anaerobic digestion of biowastes on the nitrogen fertiliser value by injection or surface banding to cereal crops

Biogas production from anaerobic digestion (AD) of biowastes is restricted by the recalcitrant nature of many substrates, and this may also reduce the fertiliser value of the produced digestate. The degradability of substrates can potentially be enhanced by physico-chemical pre-treatments before AD, and/or the degradation can be increased by a longer digestion time. In this study, we evaluated the effects of electrokinetic (high voltage) and ultrasonication pre-treatments of biowastes in a two-step AD process on nitrogen fertiliser replacement value (NFRV) of digestates obtained from two biogas plants with contrasting hydraulic retention time (HRT) in the primary AD step. The fertiliser value was tested by direct injection to spring barley and surface-banding to winter wheat, and the ammonium N was 15N-labelled to evaluate ammonia losses. The electrokinetic pre-treatment step significantly (p < 0.05) increased the NH4+-N/total N in the digestates before the second AD step but had an insignificant effect on the fertiliser value in winter wheat and spring barley. Ultrasonication pre-treatment had also no significant effect on the fertiliser value. The two-step AD significantly (p < 0.001) increased 15N recoveries and mineral fertiliser equivalence of labelled ammonium-N in winter wheat and reduced ammonia losses, with a significant effect (p < 0.001) observed in digestates sourced from a shorter HRT biogas reactor. The fertiliser equivalence of labelled ammonium-N in the digestates was 80–88% after injection, indicating relatively low N immobilisation with all the digestates. NFRV in the crops was mainly explained by the NH4+-N/total N ratio, C/N ratio and dry matter content of the digestates. The findings suggest that electrokinetic and ultrasonication pre-treatments combined with a second AD step have no considerable impact on the fertiliser value of digestates, whereas a second AD step significantly reduced ammonia losses after application by surface-banding in winter wheat.

The effect of gender on end-user preferences for yam quality descriptors.

Improving the effectiveness of breeding programs could be achieved by breeding for consumers' preferences. The relationship between gender and consumer preferences for yam was studied using purposeful sampling for key informants, focus group discussions (FGDs), and individual interviews. Data from qualitative interviews were transcribed and quantitative data analyzed using SPSS (version 20). Cramer's V (Phi) and logistic regressions were used to explain the observations. Males prioritized income-generating varieties over females, while females ranked tuber morphological characteristics high. Sex-based preference for yams showed that moldability and sweet taste are preferred by both females and males. Moderately hard boiled yams and shelf stable food products are liked by males, whereas aroma is rated highly by females. Males described yams that have a longer digestion time as "heavy" and preferred such varieties. Watery tubers and tubers not turning brown are important characteristics during processing. The Phi of 0.157 and 0.163 for sex and ethnic groups show moderate to strong associations between choices for descriptors by females, males, and various ethnic groups. These findings imply that gender disaggregation of information on preference for yam quality descriptors should be considered in future yam breeding programs as it may improve adoption rate and enhance food security.

Reaction behavior of quartz in gibbsite-boehmite bauxite in Bayer digestion and its effect on caustic consumption and alumina recovery

A high content of quartz is usually present in Australian gibbsite-boehmite bauxite. The reaction between quartz and sodium aluminate solution at high temperatures in the Bayer process can lead to loss of alumina and sodium oxide. Therefore, to improve alumina recovery, the reaction of quartz needs to be avoided. The digestion behavior of Australian gibbsite-boehmite bauxite and pure quartz in the Bayer process at 230–250 °C was systematically studied in this paper. The mineral composition and morphology of the reaction products were characterized and the kinetics of the quartz dissolution process was studied in detail. It was shown that boehmite in gibbsite-boehmite bauxite can be completely digested at high temperature (250 °C) with a short digestion time (5 min). A short digestion time results in a low reaction rate of quartz in bauxite, and is ideal for alumina recovery at high temperatures. The quartz reaction rate rapidly increases with longer digestion times. The apparent activation energy of the dissolution of quartz in bauxite in the caustic solution is 151.9 kJ mol−1, and the rate-controlling step of this reaction process is the interfacial chemical reaction. By controlling the particle size of bauxite, the digestion temperature, and the digestion time, the reaction rate of quartz in bauxite can be inhibited, which is beneficial for improving alumina recovery and reducing caustic consumption. Therefore, based on the above theoretical research, a process for digesting gibbsite-boehmite bauxite is proposed using high digestion temperature (250 °C), short digestion time (5 min) and large mineral size. An economic benefit of about US$101.9 million for a refinery with the annual output of 2 million tons of alumina can be created by the proposed process.

Factors affecting DNA barcoding classification accuracy for piscine prey: An experimental assessment with invasive lionfish

The use of DNA barcoding in fish diet studies is becoming more widespread, but the effect of prey digestion on barcoding accuracy has been poorly studied. We conducted a series of controlled feeding experiments, with red lionfish (Pterois volitans) as predators and Gulf killifish (Fundulus grandis) as prey, designed to test the effect of digestion time (12–42 h), prey item (sample) preservation (inside versus outside stomach), and sample preparation (washed versus unwashed) on DNA barcoding classification accuracy. Competitive interactions occurred between lionfish and killifish DNA during PCR amplification when universal COI primers were utilized; thus, primers were designed that matched the same priming site in both killifish and lionfish DNA. The proportion of amplified killifish DNA relative to lionfish DNA per sample was estimated using multiplex quantitative PCR (qPCR) and shown to decrease with longer digestion times, especially when samples were retained within lionfish stomachs for preservation. The effects of digestion and preservation method on amplifiable DNA resulted in significant effects on barcoding accuracy, resulting in erroneous identification of the predator (i.e., self-DNA) for nearly 25% of samples. Removing prey from predator stomachs prior to preservation significantly increased the proportion of amplified killifish DNA from samples, and enhanced barcoding accuracy. Overall, study results have important implications for the probability of false negatives for prey items, as well as false positives for self-DNA, when utilizing barcoding to characterize piscine diet. Results also highlight the need to conduct more method validation research given the increasing prevalence of DNA barcoding as a means to identify specific prey items in fish diet studies.

Porcine Lung-Derived Extracellular Matrix Hydrogel Properties Are Dependent on Pepsin Digestion Time.

Hydrogels derived from decellularized lungs are promising materials for tissue engineering in the development of clinical therapies and for modeling the lung extracellular matrix (ECM) in vitro. Characterizing and controlling the resulting physical, biochemical, mechanical, and biologic properties of decellularized ECM (dECM) after enzymatic solubilization and gelation are thus of key interest. As the role of enzymatic pepsin digestion in effecting these properties has been understudied, we investigated the digestion time-dependency on key parameters of the resulting ECM hydrogel. Using resolubilized, homogenized decellularized pig lung dECM as a model system, significant time-dependent changes in protein concentration, turbidity, and gelation potential were found to occur between the 4 and 24 h digestion time points, and plateauing with longer digestion times. These results correlated with qualitative scanning electron microscopy images and quantitative analysis of hydrogel interconnectivity and average fiber diameter. Interestingly, the time-dependent changes in the storage modulus tracked with the hydrogel interconnectivity results, while the Young's modulus values were more closely related to average fiber size at each time point. The structural and biochemical alterations correlated with significant changes in metabolic activity of several representative lung cells seeded onto the hydrogels with progressive decreases in cell viability and alterations in morphology observed in cells cultured on hydrogels produced with dECM digested for >12 and up to 72 h of digestion. These studies demonstrate that 12 h pepsin digest of pig lung dECM provides an optimal balance between desirable physical ECM hydrogel properties and effects on lung cell behaviors.

Physical, Mechanical, and Bioactive Properties of Pig Lung-Derived ECM Hydrogels are Dependent on the Duration of Pepsin Digestion

Extracellular matrix (ECM) hydrogels derived from decellularized lungs are promising materials for tissue engineering, in the development of clinical therapies, and for modeling the lung ECM in vitro. Characterizing and controlling the resulting physical, biochemical, mechanical, and biologic properties of decellularized ECM after enzymatic solubilization and gelation is thus of key interest. As the role of enzymatic pepsin digestion in effecting these properties has been understudied, we investigated the digestion time-dependency on key parameters of the resulting ECM hydrogel. Using resolubilized homogenized decellularized pig lung ECM (dECM) as a model system, significant time-dependent alterations in the protein concentration, turbidity, and gelation potential occurred between 4 and 24 hours of digestion, plateauing at longer digestion times. These results correlated with qualitative SEM images and quantitative analysis of hydrogel interconnectivity and average fiber diameter. Interestingly, the time-dependent changes in the storage modulus tracked with the hydrogel interconnectivity results, while the Young's modulus values were more closely related to average fiber size at each time point. The structural and biochemical alterations translated to significant changes in metabolic activity of several representative lung cells seeded onto the hydrogels with progressive decreases in cell viability and alterations in morphology observed in cells cultured on hydrogels produced with dECM digested for greater than 12 and up to 72 hours of digestion. These studies demonstrate that 12hr structured pepsin digest of pig lung dECM provides an optimal balance between desirable physical dECM hydrogel properties and effects on lung cell behaviors.

Incorporation of fluorophosphate in apatite

The fluorophosphate ion has been widely used as a source of fluoride in dentifrices because of its anticaries properties. Because it is isoelectronic with sulfate, which is known to replace phosphate in apatite, fluorophosphate could be expected to react similarly. In an attempt to incorporate the fluorophosphate ion into hydroxylapatite, calcium and strontium hydroxylapatites were synthesized in both aqueous and nonaqueous solutions in the presence of fluorophosphate. The corresponding fluorophosphate salts (CaFPO3 and SrFPO3) were also synthesized and found to decompose over time to their corresponding fluorapatites. Samples were characterized using solid state 19F NMR, percent fluorine analysis, and unit cell parameter analysis. Hydrolysis of the fluorophosphate ion in various solutions was determined using solution 19F NMR. The presence of hydroxylapatite or calcium ions in an aqueous solution of fluorophosphate at 80 °C and pH 7 or 9 resulted in significant hydrolysis. Inclusion of Na2FPO3 as a reactant, along with calcium or strontium nitrate and sodium phosphate, produced only fluorapatite, a result of the hydrolysis of the FPO32− ion. With a larger ratio of fluorophosphate to phosphate and longer digestion times, the corresponding fluorophosphate salt (e.g., CaFPO3) formed in addition to the fluorapatite. Attempts to incorporate fluorophosphate into hydroxylapatite using nonaqueous solvents resulted in apatites with a larger weight percent of fluorine, but there was not sufficient evidence to conclude that the fluorophosphate ion was incorporated. The use of nonaqueous solvents, including DMSO, anisole, ethanol, several ionic liquids, 1:1 mixtures of anisole in glacial acetic acid, and 15-crown-5 ether in acetonitrile, produced apatite or fluorapatite with no evidence of incorporation of FPO3. It is convenient to explain the failure of both aqueous and nonaqueous syntheses by assuming that the fluorophosphate ion, perhaps in an unstable apatite phase, serves to provide fluoride to the apatite channel while the displaced hydroxide reacts with the fluorophosphate to form phosphate for the formation of fluorapatite.

The pros and cons of increased trypsin-to-protein ratio in targeted protein analysis

The effect of increasing the trypsin amount in bottom-up based targeted protein analysis is evaluated. By applying an increased trypsin-to-protein ratio (1:1 (w/w)) after heat denaturation (60°C), reduction and alkylation, the digestion time could be reduced profoundly compared to conventional digestion conditions (ratio 1:40, overnight) without compromising method sensitivity or digestion repeatability. The procedure was obtained after a systematic evaluation of trypsin level and trypsin quality using a set of three model proteins: human chorionic gonadotropin (hCG), bovine serum albumin (BSA) and cytochrome C (CytC). All peptides monitored were produced at similar or higher levels after 45min at trypsin-to-protein ratio 1:1, compared to conventional overnight digestion (exception: CytC using modified trypsin, required up to 4h (at 1:1 ratio) in order achieve this). Peptide decay due to chymotryptic activity was observed at longer digestion times, but the effect was circumvented using digestion times <4h. The accelerated digestion protocol (1:1 (w/w), 45min) was applied to realistic human serum samples containing the biomarker protein hCG to demonstrate its applicability.

Alkaline treatment of high-solids sludge and its application to anaerobic digestion.

High-solids anaerobic digestion is a promising new process for sludge reduction and bioenergy recovery, requiring smaller digestion tanks and less energy for heating, but a longer digestion time, than traditional low-solids anaerobic digestion. To accelerate this process, alkaline sludge disintegration was tested as a pretreatment method for anaerobic digestion of high-solids sludge. The results showed that alkaline treatment effectively disintegrated both low-solids sludge and high-solids sludge, and treatment duration of 30 min was the most efficient. The relation between sludge disintegration degree and NaOH dose can be described by a transmutative power function model. At NaOH dose lower than 0.2 mol/L, sludge disintegration degree remained virtually unchanged when sludge total solids (TS) content increased from 2.0 to 11.0%, and decreased only slightly when sludge TS increased to 14.2%. Although high-solids sludge required a slightly higher molarity of NaOH to reach the same disintegration level of low-solids sludge, the required mass of NaOH actually decreased due to sludge thickening. From the view of NaOH consumption, sludge TS of 8-12% and a NaOH dose of 0.05 mol/L were optimum conditions for alkaline pretreatment, which resulted in a slight increase in accumulative biogas yield, but a decrease by 24-29% in digestion time during the subsequent anaerobic digestion.

Effects of enzymatic treatments on the depth-dependent viscoelastic shear properties of articular cartilage.

Osteoarthritis (OA) is a disease that involves the erosion and structural weakening of articular cartilage. OA is characterized by the degradation of collagen and proteoglycans in the extracellular matrix (ECM), particularly at the articular surface by proteinases including matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs).(1) Degradation of collagen and proteoglycans is known to alter shear mechanical properties of cartilage, but study of this phenomenon has been focused on bulk tissue properties. The purpose of this study was to assess microscale cartilage damage induced by trypsin or collagenase using a technique to measure the local shear viscoelastic properties. Safranin-O histology revealed a decrease in proteoglycans near the articular surface after collagenase and trypsin digestions, with proteoglycan depletion increasing in time. Similarly, confocal reflectance micrographs showed increasing collagen degradation in collagenase treated samples, although the collagen network remained intact after trypsin treatment. Both treatments induced changes in shear modulus that were confined to a narrow range (∼400µm) near tissue surface. In addition, collagenase altered the total energy dissipation distribution by up to a factor of 100, with longer digestion times corresponding to higher energy dissipation. The ability to detect local mechanical signatures in tissue composition and mechanics is an important tool for understanding the spatially non-uniform changes that occur in articular cartilage diseases such as OA.

Determination of the extraction kinetics for the quantification of polyhydroxyalkanoate monomers in mixed microbial systems

For the first time, a systematic approach was conducted to determine the key factors influencing the kinetics of hydroxyalkanote (HA) extraction in biological systems. Six mixed microbial systems where polyhydroxyalkanoate (PHA) is produced were evaluated. Experiments were carried out for full-scale and lab-scale activated sludge systems using different configurations (containing floccular or granular sludge), as well as specific PHA accumulating cultures that contain high or low intracellular PHA fractions. The overall reaction was limited by the kinetics of the PHA hydrolysis in floccular cultures, whereas in granular cultures, it was limited by the cell lysis step. The monomeric composition of the polymer also had an impact on the HA extraction rate: higher acid concentration and a longer digestion time should be employed when cells accumulate monomers with more substituents, such as hydroxy-2-methylbutyrate (H2MB) and hydroxy-2-methylvalerate (H2MV). This study optimised the method for HA extraction, which impacts the assessment of the quantity and quality of PHA biopolymers.

A Systematic Reevaluation of Enzymatic Isolation Methodology for Nucleus Pulposus Cells

IntroductionThe nucleus pulposus (NP) is the central part of IVD and understanding its function is essential for studying IVD degeneration. Enzymatic digestion by collagenase is used to release cel...

Effects of alkaline or liquid-ammonia treatment on crystalline cellulose: changes in crystalline structure and effects on enzymatic digestibility

BackgroundIn converting biomass to bioethanol, pretreatment is a key step intended to render cellulose more amenable and accessible to cellulase enzymes and thus increase glucose yields. In this study, four cellulose samples with different degrees of polymerization and crystallinity indexes were subjected to aqueous sodium hydroxide and anhydrous liquid ammonia treatments. The effects of the treatments on cellulose crystalline structure were studied, in addition to the effects on the digestibility of the celluloses by a cellulase complex.ResultsFrom X-ray diffractograms and nuclear magnetic resonance spectra, it was revealed that treatment with liquid ammonia produced the cellulose IIII allomorph; however, crystallinity depended on treatment conditions. Treatment at a low temperature (25°C) resulted in a less crystalline product, whereas treatment at elevated temperatures (130°C or 140°C) gave a more crystalline product. Treatment of cellulose I with aqueous sodium hydroxide (16.5 percent by weight) resulted in formation of cellulose II, but also produced a much less crystalline cellulose. The relative digestibilities of the different cellulose allomorphs were tested by exposing the treated and untreated cellulose samples to a commercial enzyme mixture (Genencor-Danisco; GC 220). The digestibility results showed that the starting cellulose I samples were the least digestible (except for corn stover cellulose, which had a high amorphous content). Treatment with sodium hydroxide produced the most digestible cellulose, followed by treatment with liquid ammonia at a low temperature. Factor analysis indicated that initial rates of digestion (up to 24 hours) were most strongly correlated with amorphous content. Correlation of allomorph type with digestibility was weak, but was strongest with cellulose conversion at later times. The cellulose IIII samples produced at higher temperatures had comparable crystallinities to the initial cellulose I samples, but achieved higher levels of cellulose conversion, at longer digestion times.ConclusionsEarlier studies have focused on determining which cellulose allomorph is the most digestible. In this study we have found that the chemical treatments to produce different allomorphs also changed the crystallinity of the cellulose, and this had a significant effect on the digestibility of the substrate. When determining the relative digestibilities of different cellulose allomorphs it is essential to also consider the relative crystallinities of the celluloses being tested.

Detrimental effect of excessive collagenase class II on human islet isolation outcome

Commercially available purified collagenases, derived from Clostridium histolyticum, contain two different classes of collagenase: class I collagenase (CI) and class II collagenase (CII) at a predetermined ratio. In this study, using purified CI and CII in separate vials, we had a unique opportunity to investigate the effect of the proportion between two collagenase classes on clinical human islet isolation. Pancreas organs derived from deceased donors were prospectively assigned to one of three different enzyme protocols: group A--CII:CI = 1:1 vial; group B--1:2; group C--1.5:1. As a result, their total collagenase activities were 2116, 2230, and 3117 Wunsch units/pancreas in groups A, B, and C, respectively but thermolysin dosage was adjusted to 624-988 Units/g pancreas. The pancreas was not efficiently digested in group C in spite of a relatively longer digestion time and significantly higher Wunsch activity, resulting in the poorest islet isolation outcome among the three groups. Additional retrospective analysis revealed that this suboptimal outcome in group C was not because of the absolute excessive amount of collagenase activity but as a result of the relatively high proportion of CII (i.e., unbalanced CII/CI ratio). Our study suggests that an excessive CII is ineffective in releasing islets from human pancreas, and rather a balanced CII/CI ratio is of paramount importance.

Factors That Affect Human Islet Isolation

More than 10,000 IEQ/kg recipient weight of islets is often necessary to achieve insulin independence in patients with type 1 diabetes mellitus. Several studies have identified high donor body mass index (BMI) and pancreas size as important factors for the success of human islet isolation. However, the donor shortage underscores the need to improve isolation outcomes from lower BMI pancreas donors and/or small pancreata. The aim of this study was to identify the critical factors that affect isolation outcome. We analyzed the data from 207 isolations performed from 2002 to 2006 with respect to donor characteristics, pancreas condition, and processing variables. More than 3000 IEQ/g pancreas weight was considered to be an acceptable isolation outcome. This goal was obtained from donors with a BMI >30 kg/m 2 ( P = .002). The pancreatic surface integrity was also a significant factor ( P = .02). Moreover, longer digestion times ( P = .04) and a greater proportion of trapped islets negatively affected success rates ( P = .004). As previously reported, pancreata from high BMI donors were suitable for islet isolation and transplantation, as they yielded higher total islet particle numbers and higher IEQ/g. Although BMI and pancreas size are not controllable due to the organ donor shortage, factors such as pancreatic surface integrity, shorter digestion time, and lower proportions of trapped islets were found to be significant to obtain higher success rates. The development of better protocols and systematic training of processing/procurement teams will be of assistance to increase the number of successful human islet isolations.

Diel feeding pattern of whiting in the North Sea

Though numerous studies have analysed the feeding periodicity of North Sea gadoids, no general diel pattern has been found. The lack of agreement between studies may be related to differences in prey composition and behaviour, but it has not been attempted to link the diel intake pattern directly to intake of individual prey. This study presents an analysis of the round the clock occurrence of several prey types in the stomachs of whiting Merlangius merlangus, a major predator on fish and crustaceans in the North Sea. Generalised linear models were used to determine if the occurrence of different prey varied significantly with time of day and whether this diel pattern dif- fered between locations and predator size groups. The results show that the occurrence of bottom dwelling prey increased significantly during the night at 4 of 5 locations. In contrast, free swimming prey and prey migrating towards the demersal layer during the day were eaten mainly in the daylight hours. No diel pattern in the presence of larger fish prey could be found, presumably due to their longer digestion time. A general diel pattern in catch rates of the predator could not be detected and the analyses did not appear to be biased by vertical migration of the predator. The results have important implications for the understanding of prey selection by wild predators, as this is, in effect, a choice between temporally co-occurring prey.