Cryo-EM Structure of the Fork Protection Complex Bound to CMG at a Replication Fork.

The eukaryotic replisome, organized around the Cdc45-MCM-GINS (CMG) helicase, orchestrates chromosome replication. Multiple factors associate directly with CMG, including Ctf4 and the heterotrimeric fork protection complex (Csm3/Tof1 and Mrc1), which has important roles including aiding normal replication rates and stabilizing stalled forks. How these proteins interface with CMG to execute these functions is poorly understood. Here we present 3 to 3.5 Å resolution electron cryomicroscopy (cryo-EM) structures comprising CMG, Ctf4, and the fork protection complex at a replication fork. The structures provide high-resolution views of CMG-DNA interactions, revealing a mechanism for strand separation, and show Csm3/Tof1 "grip" duplex DNA ahead of CMG via a network of interactions important for efficient replication fork pausing. Although Mrc1 was not resolved in our structures, we determine its topology in the replisome by cross-linking mass spectrometry. Collectively, our work reveals how four highly conserved replisome components collaborate with CMG to facilitate replisome progression and maintain genome stability.

A conserved mechanism for regulating replisome disassembly in eukaryotes.

Replisome disassembly is the final step of eukaryotic DNA replication and is triggered by ubiquitylation of the CDC45-MCM-GINS (CMG) replicative helicase1-3. Despite being driven by evolutionarily diverse E3 ubiquitin ligases in different eukaryotes (SCFDia2 in budding yeast1, CUL2LRR1 in metazoa4-7), replisome disassembly is governed by a common regulatory principle, in which ubiquitylation of CMG is suppressed before replication termination, to prevent replication fork collapse. Recent evidence suggests that this suppression is mediated by replication fork DNA8-10. However, it is unknown how SCFDia2 and CUL2LRR1 discriminate terminated from elongating replisomes, to selectively ubiquitylate CMG only after termination. Here we used cryo-electron microscopy to solve high-resolution structures of budding yeast and human replisome-E3 ligase assemblies. Our structures show that the leucine-rich repeat domains of Dia2 and LRR1 are structurally distinct, but bind to a common site on CMG, including the MCM3 and MCM5 zinc-finger domains. The LRR-MCM interaction is essential for replisome disassembly and, crucially, is occluded by the excluded DNA strand at replication forks, establishing the structural basis for the suppression of CMG ubiquitylation before termination. Our results elucidate a conserved mechanism for the regulation of replisome disassembly in eukaryotes, and reveal a previously unanticipated role for DNA in preserving replisome integrity.

Structural mechanisms of SLF1 interactions with Histone H4 and RAD18 at the stalled replication fork.

DNA damage that obstructs the replication machinery poses a significant threat to genome stability. Replication-coupled repair mechanisms safeguard stalled replication forks by coordinating proteins involved in the DNA damage response (DDR) and replication. SLF1 (SMC5-SMC6 complex localization factor 1) is crucial for facilitating the recruitment of the SMC5/6 complex to damage sites through interactions with SLF2, RAD18, and nucleosomes. However, the structural mechanisms of SLF1's interactions are unclear. In this study, we determined the crystal structure of SLF1's ankyrin repeat domain bound to an unmethylated histone H4 tail, illustrating how SLF1 reads nascent nucleosomes. Using structure-based mutagenesis, we confirmed a phosphorylation-dependent interaction necessary for a stable complex between SLF1's tandem BRCA1 C-Terminal domain (tBRCT) and the phosphorylated C-terminal region (S442 and S444) of RAD18. We validated a functional role of conserved phosphate-binding residues in SLF1, and hydrophobic residues in RAD18 that are adjacent to phosphorylation sites, both of which contribute to the strong interaction. Interestingly, we discovered a DNA-binding property of this RAD18-binding interface, providing an additional domain of SLF1 to enhance binding to nucleosomes. Our results provide critical structural insights into SLF1's interactions with post-replicative chromatin and phosphorylation-dependent DDR signalling, enhancing our understanding of SMC5/6 recruitment and/or activity during replication-coupled DNA repair.

Characterization of recombinant phytase of Klebsiella sp. and the influence of novel 3-phytase on mineral solubility in broiler diets under an in vitro digestion assay.

Phytate (inositol hexaphosphate) is the major storage form of phosphorus (P) in nature, and phytases catalyze the hydrolysis of P from phytate and the formation of inositol phosphate isomers. In this study, a bacterium that produces phytase was isolated in a phytase screening medium. The bacterium was identified as Klebsiella sp. using phenotypic and molecular techniques. The PhyK phytase gene was successfully amplified from the genome, inserted into the pET-21a (+) vector, and expressed as a recombinant protein in E. Coli BL21. The efficiency of a laboratory phytase (Lab-Ph, PhyK phytase) was determined and compared with a commercial phytase (Com-Ph, Quantum Blue 40P phytase, AB Vista) under an in vitro digestion assay. The native signal peptide effectively facilitated the translocation of the protein to the periplasmic space of E. Coli BL21, resulting in the proper folding of the protein and the manifestation of desirable enzyme activity. The Lab-Ph displayed the temperature and pH optima at 50 °C and 5 respectively. In addition, the Lab-Ph was inactivated at 80 °C. Under an in vitro digestion assay condition, Lab-Ph improved the P solubility coefficient in broiler diets. In comparison, the Com-Ph significantly increased the P solubility coefficient even when compared with the Lab-Ph. In summary, this study has shown that Lab-Ph possesses the necessary biochemical properties to be used in various industrial applications. However, Lab-Ph is extremely sensitive to heat treatment. The Lab-Ph and Com-Ph under an in vitro digestion assay improved the solubility coefficient of P in the broiler diet.

The role of feed enzymes in maintaining poultry intestinal health.

Gut health or intestinal health is frequently discussed without any clear definition as to its meaning. It is suggested that this should be defined as intestinal integrity and functionality as both are a pre-requisite for the health of the intestine itself and the host. The health of the intestine is dependent upon a successful evolution of the absorptive capacity of the intestine, which in turn is influenced by the co-evolution of the intestinal immune systems and the microbiota. Nutrient supply plays a significant role in this process and from the perspective of the microbiota this changes with age as the intestines and upper gastrointestinal tract (GIT) microbiota become more effective in nutrient removal. Feed enzymes play a significant role in this process. Phytases can improve digestion of minerals, amino acids and energy and as a result reduce the availability of nutrients in the lower intestines for the microbiota. Protease can have a similar effect with amino acid supply. Non-starch polysaccharidases (NSPases) have a unique role in that they not only improve diet digestibility from the hosts perspective, thus limiting nutrient supply to the microbiota, but they also release soluble fragments of fibre from the insoluble matrix and/or depolymerize high molecular weight viscous fibre fractions in to smaller, more fermentable carbohydrate fractions. This results in a more favourable balance between fermentable carbohydrate to protein supply, a ratio which is deemed critical to maintaining good intestinal health. The dynamic nature of this complex evolution needs greater consideration if antibiotic free production is to succeed. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

SnoopLigase Catalyzes Peptide-Peptide Locking and Enables Solid-Phase Conjugate Isolation.

Simple, efficient reactions for connecting biological building-blocks open up many new possibilities. Here we have designed SnoopLigase, a protein that catalyzes site-specific transamidation, forming an isopeptide bond with more than 95% efficiency between two peptide tags, SnoopTagJr and DogTag. We initially developed these components by three-part splitting of the Streptococcus pneumoniae adhesin RrgA. The units were then engineered, guided by structure, bioinformatic analysis of sequence homology, and computational prediction of stability. After engineering, SnoopLigase demonstrated high-yield coupling under a wide range of buffers and temperatures. SnoopTagJr and DogTag were functional at the N- or C-terminus, while DogTag was also functional at internal sites in proteins. Having directed reaction of SnoopTagJr and DogTag, SnoopLigase remained stably bound to the ligated product, thus reconstituting the parent domain. Separating products from unreacted starting material and catalyst is often as challenging as reactions themselves. However, solid-phase immobilization of SnoopLigase enabled the ligated SnoopTagJr-DogTag product to be eluted with high purity, free from SnoopLigase or unligated substrates. The solid-phase catalyst could then be reused multiple times. In search of a generic route to improve the resilience of enzymes, we fused SnoopTagJr to the N-terminus and DogTag to the C-terminus of model enzymes, allowing cyclization via SnoopLigase. While wild-type phytase and ß-lactamase irreversibly aggregated upon heating, cyclization using SnoopLigase conferred exceptional thermoresilience, with both enzymes retaining solubility and activity following heat treatment up to 100 °C. SnoopLigase should create new opportunities for conjugation and nanoassembly, while illustrating how to harness product inhibition and extend catalyst utility.

The evolution and application of enzymes in the animal feed industry: the role of data interpretation.

1. Enzymes have been used commercially for nearly 40 years and save significant costs through sparing of expensive nutrients but the mechanism by which this is achieved is still debated. 2. The research focused on non-starch polysaccharidase (NSPase) enzymes is used as an example of where greater progress could have been made if the details of the work had been described more fully and the analysis of the data generated had been broader in scope and more critical. 3. Lack of standardisation of the details presented in the materials and methods has been identified as a significant barrier to meaningful retrospective analysis and thus limits advances in the understanding of the mode of action of these enzymes. 4. The identity of the enzyme employed and its activity is often lacking, and more importantly the purity is rarely disclosed. Contaminant activities which are neither listed nor assayed could play a significant role in the responses observed. 5. The dose optimum of most enzymes is often considerably higher than that employed in most studies. Thus studies claiming synergy between two 'activities' should ensure that the response is not related to each enzyme simply augmenting the dose of just one activity in the finished feed. This is a common problem, and coupled with the lack of factorial experiments to justify the presence of each enzyme in a multi-enzyme product, it is not surprising that there is still debate as to whether single or multi-enzymes are best suited poultry rations. 6. The three proposed mechanisms for NSPases (viscosity, cell wall and prebiotic) are discussed, and along with their strengths and weaknesses it is suggested that a re-evaluation of each is needed. Viscosity may have to be re-evaluated as being a function not only of the cereal being fed, but of the age of the animal as well. The cell wall theory as described is poorly modelled in vitro and hence the validity of these data is questioned. The prebiotic theory may need significant modification as it appears that the quantities of oligomers produced are insufficient to generate the additional volatile fatty acids (VFA)'s reported. It is likely that all three mechanisms play a role in the responses observed, but the prebiotic mechanism probably plays by far the most important part in low viscosity diets. 7. Future research would be improved if it considered all potential mechanisms when designing a trial. Significant failings are apparent as a result of adherence to tenets in explanation of the results. Most importantly, it should be emphasised that a hypothesis is there to be tested, not defended.

Older people remain on blood pressure agents despite being hypotensive resulting in increased mortality and hospital admission.

BACKGROUND: the use of antihypertensive medication in older people in order to prevent cardiovascular events is well established. The use of such agents has been encouraged by incentive schemes in the United Kingdom including the Quality and Outcomes Framework. In addition, many guidelines recommend good blood pressure (BP) control in the elderly. However, in older people antihypertensives can cause adverse effects related to hypotension. AIM: the aim of this study was to assess the prevalence of low BP and impact on outcomes, particularly in the presence of antihypertensive treatment, in a primary care population of older people. DESIGN: a retrospective observational cohort study in people over the age of 70 years registered with primary care providers in Kent. RESULTS: a total of 11,167 patients over 70 years old were analysed, 6,373 female (57%). Systolic blood pressure (SBP) was below 120 mmHg in 1,297 people (844 on antihypertensives), below 110 mmHg in 474 (313 on antihypertensives) and below 100 mmHg in 128 (89 on antihypertensives). Hypotension was independently associated with mortality, acute kidney injury and hospital admission. CONCLUSIONS: the results demonstrate that low SBP is associated with adverse events, it is possible that the pursuit of BP control at a population level may lead to over-treatment in certain groups of patients. This may result in an increased incidence of adverse events particularly in older people.

Ovodefensins, an Oviduct-Specific Antimicrobial Gene Family, Have Evolved in Birds and Reptiles to Protect the Egg by Both Sequence and Intra-Six-Cysteine Sequence Motif Spacing.

Ovodefensins are a novel beta defensin-related family of antimicrobial peptides containing conserved glycine and six cysteine residues. Originally thought to be restricted to the albumen-producing region of the avian oviduct, expression was found in chicken, turkey, duck, and zebra finch in large quantities in many parts of the oviduct, but this varied between species and between gene forms in the same species. Using new search strategies, the ovodefensin family now has 35 members, including reptiles, but no representatives outside birds and reptiles have been found. Analysis of their evolution shows that ovodefensins divide into six groups based on the intra-cysteine amino acid spacing, representing a unique mechanism alongside traditional evolution of sequence. The groups have been used to base a nomenclature for the family. Antimicrobial activity for three ovodefensins from chicken and duck was confirmed against Escherichia coli and a pathogenic E. coli strain as well as a Gram-positive organism, Staphylococcus aureus, for the first time. However, activity varied greatly between peptides, with Gallus gallus OvoDA1 being the most potent, suggesting a link with the different structures. Expression of Gallus gallus OvoDA1 (gallin) in the oviduct was increased by estrogen and progesterone and in the reproductive state. Overall, the results support the hypothesis that ovodefensins evolved to protect the egg, but they are not necessarily restricted to the egg white. Therefore, divergent motif structure and sequence present an interesting area of research for antimicrobial peptide design and understanding protection of the cleidoic egg.

The economic impact of acute kidney injury in England.

BACKGROUND: Acute kidney injury (AKI) is one of the most common complications affecting hospital inpatients around the world. It is associated with high mortality and adverse long-term outcomes, but there is uncertainty regarding its prevalence and cost. We estimate the prevalence of AKI in hospital inpatients in a universal health-care system, and the immediate and long-term impacts on survival, quality of life and health-care costs. METHODS: We examined prevalence of AKI in inpatients using both routine national data for the National Health Service (NHS) in England, and laboratory data from East Kent Hospitals. We used regression analyses to estimate the impact of AKI on mortality and length of hospital stay, and a Markov model to estimate the impact on quality-adjusted life years and NHS costs. RESULTS: AKI was recorded in 2.43% of hospital admissions in Hospital Episode Statistics (HES), but age- and gender-standardized estimates derived from laboratory data suggest the true prevalence may be more than five times as high (14.15%). We estimate that the annual number of excess inpatient deaths associated with AKI in England may be above 40,000. The annual cost of AKI-related inpatient care in England is estimated at £1.02 billion, just over 1% of the NHS budget. The lifetime cost of post-discharge care for people who had AKI during hospital admission in 2010-11 is estimated at £179 million. CONCLUSIONS: AKI prevalence in inpatients may be considerably higher than previously thought, and up to four fifths of cases may not be captured in routine hospital data. AKI is associated with large numbers of in-hospital deaths and with high NHS costs. Comparison of HES and East Kent data suggests that most of the cases recorded in HES may be relatively severe AKI (AKIN 2-3).

Black ethnicity predicts better survival on dialysis despite greater deprivation and co-morbidity: a UK study.

BACKGROUND: Observational studies from the United States have identified black race as conferring a survival advantage on dialysis. This study represents the first large single-center study from a UK dialysis unit examining the outcome of ethnic minorities on renal replacement therapy (RRT). METHODS: A retrospective analysis of all patients of white or black race initiating RRT at King's College Hospital Renal Unit, London, between 1996 and 2008 was performed. A total of 1,340 patients were studied, of which 952 (71%) were of white race, and 388 (29%) were of black race. Kaplan-Meier survival curves, the log rank test and Cox's proportional hazard models were used to compare survival between groups. RESULTS: The results revealed black ethnicity to be associated with a significant survival benefit on dialysis. This was the case even after adjustment for age, gender, diabetes, transplantation, and deprivation. In those patients not transplanted, black race conferred a hazard ratio (HR) of 0.51 (95% CI 0.41 - 0.63) over 5 years. CONCLUSIONS: This study provides evidence for a lower mortality rate amongst black patients on dialysis in comparison with their white counterparts in the UK. The reasons behind this remain poorly understood but a lower incidence of cardiovascular disease in black patients and more kidney-limited disease may be important.

Do acute elevations of serum creatinine in primary care engender an increased mortality risk?

BACKGROUND: The significant impact Acute Kidney Injury (AKI) has on patient morbidity and mortality emphasizes the need for early recognition and effective treatment. AKI presenting to or occurring during hospitalisation has been widely studied but little is known about the incidence and outcomes of patients experiencing acute elevations in serum creatinine in the primary care setting where people are not subsequently admitted to hospital. The aim of this study was to define this incidence and explore its impact on mortality. METHODS: The study cohort was identified by using hospital data bases over a six month period. INCLUSION CRITERIA: People with a serum creatinine request during the study period, 18 or over and not on renal replacement therapy.The patients were stratified by a rise in serum creatinine corresponding to the Acute Kidney Injury Network (AKIN) criteria for comparison purposes. Descriptive and survival data were then analysed.Ethical approval was granted from National Research Ethics Service (NRES) Committee South East Coast and from the National Information Governance Board. RESULTS: The total study population was 61,432. 57,300 subjects with 'no AKI', mean age 64.The number (mean age) of acute serum creatinine rises overall were, 'AKI 1' 3,798 (72), 'AKI 2' 232 (73), and 'AKI 3' 102 (68) which equates to an overall incidence of 14,192 pmp/year (adult). Unadjusted 30 day survival was 99.9% in subjects with 'no AKI', compared to 98.6%, 90.1% and 82.3% in those with 'AKI 1', 'AKI 2' and 'AKI 3' respectively. After multivariable analysis adjusting for age, gender, baseline kidney function and co-morbidity the odds ratio of 30 day mortality was 5.3 (95% CI 3.6, 7.7), 36.8 (95% CI 21.6, 62.7) and 123 (95% CI 64.8, 235) respectively, compared to those without acute serum creatinine rises as defined. CONCLUSIONS: People who develop acute elevations of serum creatinine in primary care without being admitted to hospital have significantly worse outcomes than those with stable kidney function.

What is the real impact of acute kidney injury?

BACKGROUND: Acute kidney injury (AKI) is a common clinical problem. Studies have documented the incidence of AKI in a variety of populations but to date we do not believe the real incidence of AKI has been accurately documented in a district general hospital setting.The aim here was to describe the detected incidence of AKI in a typical general hospital setting in an unselected population, and describe associated short and long-term outcomes. METHODS: A retrospective observational database study from secondary care in East Kent (adult catchment population of 582,300). All adult patients (18 years or over) admitted between 1st February 2009 and 31st July 2009, were included. Patients receiving chronic renal replacement therapy (RRT), maternity and day case admissions were excluded. AKI was defined by the acute kidney injury network (AKIN) criteria. A time dependent risk analysis with logistic regression and Cox regression was used for the analysis of in-hospital mortality and survival. RESULTS: The incidence of AKI in the 6 month period was 15,325 pmp/yr (adults) (69% AKIN1, 18% AKIN2 and 13% AKIN3). In-hospital mortality, length of stay and ITU utilisation all increased with severity of AKI. Patients with AKI had an increase in care on discharge and an increase in hospital readmission within 30 days. CONCLUSIONS: This data comes closer to the real incidence and outcomes of AKI managed in-hospital than any study published in the literature to date. Fifteen percent of all admissions sustained an episode of AKI with increased subsequent short and long term morbidity and mortality, even in those with AKIN1. This confers an increased burden and cost to the healthcare economy, which can now be quantified. These results will furnish a baseline for quality improvement projects aimed at early identification, improved management, and where possible prevention, of AKI.

Basis for the diversity and extent in loss of digestible nutrients created by dietary phytin: Emphasis on fowl and swine.

Phytin is the Ca2+-Mg2+-K+ salt of phytic acid that is created and deposited in the aleurone layer and/or germ of grains and legumes. Its high presence in feedstuffs for fowl and swine diets results in it being a universal and significant impediment to optimum performance. Phytin impairs gastrointestinal recovery of a wide array of nutrients, the effect varying with the nutrient concerned. On exposure to low pH during gastric digestion, phytin dissociates into phytic acid and solubilized Ca2+. Even at low gastric pH, phytic acid is negatively charged which forms the basis of its anti-nutritive behavior. Pepsinogen has extensive basic amino acids on its activation peptide that are presented as cations at low pH which are targeted by pepsin for activation. Partially crystalized Ca2+ near the enzyme's active site further stabilizes its newly formed structure. Thus, phytic acid appears to interfere with gastric digestion by several mechanisms; interfering with pepsinogen activation by binding to the polypeptide's basic amino acids; coordinating free Ca2+, destabilizing pepsin; binding some dietary proteins directly, further compromising gastric proteolysis. Upon digesta attaining neutrality in the duodenum, Ca2+ and other cations re-bind with accessible anions, phytic acid being a significant contender. Phytate not only binds free cations but can also strip them from enzymes (e.g. Ca2+, Zn2+) which reduces their structural resistance to autolysis and ability as co-factors (e.g. Zn2+) to increase enzyme activity. Goblet cells initially employ Ca2+ as an electronic shield between mucin layers enabling granule formation and cell storage. After mucin granule release, Ca2+ is progressively displaced by Na+ to free the viscous mucins enabling its translocation. Mucin entangles with the glycocalyx of adjacent enterocytes thereby constructing the unstirred water layer (USWL). Excessive removal of Ca2+ from mucin by phytic acid increases its fluidity facilitating its loss from the USWL with its associated Na+. This partly explains increased mucin and Na+ losses noted with high phytate diets. This review suggests that phytic acid binding of Ca2+ and less so Zn2+ is the basis for the diversity in nutrient losses encountered and that such losses are in proportion to dietary phytate content.

Dietary fibre effects and the interplay with exogenous carbohydrases in poultry nutrition.

A comprehensive understanding of the role of dietary fibre in non-ruminant animal production is elusive. Equivocal and conflated definitions of fibre coupled with significant analytical complexity, interact with poorly defined host and microbiome relationships. Dietary fibre is known to influence gut development, feed intake and passage rate, nutrient absorption, microbiome taxonomy and function, gut pH, endogenous nutrient loss, environmental sustainability, animal welfare and more. Whilst significant gaps persist in our understanding of fibre in non-ruminant animal production, there is substantial interest in optimizing the fibre fraction of feed to induce high value phenotypes such as improved welfare, live performance and to reduce the environmental footprint of animal production systems. In order to achieve these aspirational goals, it is important to tackle dietary fibre with the same level of scrutiny as is currently done for other critical nutrient classes such as protein, minerals and vitamins. The chemical, mechanical and nutritional role of fibre must be explored at the level of monomeric sugars, oligosaccharides and polysaccharides of varying molecular weight and decoration, and this must be in parallel to standardisation of analytical tools and definitions for speciation. To further complicate subject, exogenous carbohydrases recognise dietary fibre as a focal substrate and have varying capacity to generate lower molecular weight carbohydrates that interact differentially with the host and the enteric microbiome. This short review article will explore the interactive space between dietary fibre and exogenous carbohydrases and will include their nutritional and health effects with emphasis on functional development of the gut, microbiome modulation and host metabolism.

Interactive effects of a stimbiotic supplementation and wheat bran inclusion in corn- or wheat-based diets on growth performance, ileal digestibility, and expression of nutrient transporters of broilers chickens.

A 42-day experiment was conducted to investigate the interactive effects of a stimbiotic (STB) and wheat bran (WB) in broiler chickens receiving diets (DT) based on corn or wheat. A total of 960 Cobb 500 male broiler chicks at zero-day old were allocated to 64 pens with 8 treatments, 8 replicates per treatment, and 15 birds per replicate. The treatments were arranged in a randomized complete block design with 2 × 2 × 2 factorial, with the factors as diet (corn-soybean meal or wheat-soybean meal), STB (with or without), and WB (0 or 50 g/kg). Body weight gain (BWG), feed intake (FI), and mortality-corrected FCR data were collected for the starter (d 0-10), grower (d 10-28), and finisher (d 28-42) phases. Ileal digesta and jejunal tissue were collected on d 18 and 42. Data were analyzed as a 2 × 2 × 2 factorial using a mixed model of JMP. There was no significant 3-way interaction for growth performance and expression of nutrient transporters. There was a significant SB × WB for FCR (P < 0.05) in the grower phase. Stimbiotic supplementation and WB inclusion individually improved (P < 0.05) FCR regardless of cereal type, but this effect was not observed when the two were combined. In the overall phase (d 0-42), birds receiving corn-based diets had higher (P < 0.05) FI and BWG than those receiving wheat-based diets. There was a significant 3-way interaction for ileal digestible energy (IDE) (P < 0.05) and N digestibility (ND) on d 18 and 42 (P < 0.05). There was no effect of STB or WB in corn-based diets, whereas WB inclusion decreased IDE in wheat-based diets, but STB supplementation increased IDE with or without WB. In corn-based diets, WB inclusion increased ND, whereas ND was decreased in wheat-based diets. Supplementation with STB had no effect on ND, but STB increased ND in wheat-based diets with and without WB. Ileal DE was greater (P < 0.05) for wheat- than corn-based diets on d 42. Wheat bran inclusion increased ND in corn-based diets whereas, with STB supplementation, there was no difference in ND with or without WB. There was no difference in ND with or without STB or WB in wheat-based diets. There was a significant STB × DT on the expression of GLUT-1 (P < 0.05). In corn-based diets, STB produced an upward expression of GLUT-1, whereas in wheat-based diets, STB supplementation had no effect. On d 42, stimbiotic supplementation produced upward (P < 0.05) expression of SGLT-1. In conclusion, STB supplementation in wheat- or corn-based diets and with WB inclusion improved energy digestibility. On the other hand, WB inclusion in wheat-based diets decreased nutrient digestibility especially on d 18, thus making room for a positive response to STB supplementation in wheat-based diets compared to the marginal response to STB supplementation observed in corn-based diets.

Unveiling the influence of adaptation time on xylanase and arabinoxylan-oligosaccharide efficacy: a study on nutrient digestibility, viscosity, and scanning electron microscopy in the small and large intestine of growing pigs fed insoluble fiber.

The experiment objective was to evaluate the impact of xylanase over time on viscosity and digestibility in growing pigs fed corn-based fiber. Twenty gilts with an initial body weight of 30.6 ±â€…0.2 kg (n = 5 per dietary treatment) were fitted with t-cannulae in the medial jejunum and terminal ileum, housed individually, and randomly assigned to one of four dietary treatments: low-fiber control (LF) with 10.4% total dietary fiber (TDF), 30% corn bran high-fiber control (HF; 26.4% TDF), HF + 100 mg xylanase/kg (XY; Econase XT 25P; AB Vista, Marlborough, UK), and HF + 50 mg arabinoxylan-oligosaccharide/kg (AX). Gilts were limit fed for three 17 d periods (P1, P2, P3); each included 5 d adaptation, 2 d fecal collection, 3 d ileal collection, 3 d jejunal collection, and 4 d related rate of passage study. Data were analyzed as repeated measures using a linear mixed model with surgery date as a random effect, and dietary treatment, period, and their interaction as fixed effects. Jejunal and ileal digesta viscosity did not differ among dietary treatments or periods (P > 0.10). There was a dietary treatment × period interaction for the apparent jejunal digestibility (AJD) of dry matter (DM), gross energy (GE), insoluble dietary fiber (IDF), neutral detergent fiber (NDF), total arabinoxylan (T-AX), total non-starch polysaccharide (T-NSP), and TDF (P≤ 0.05). In P1, LF had the greatest AJD of DM (15.5%), and relative to HF and AX, XY decreased it (9.3%, 10.1 %, and 6.3%, respectively). In P2, the AJD of DM in XY was greater than HF (11.7% vs. 9.1%) but did not differ from AX (10.5%). Relative to HF, in P3, XY increased AJD of DM (11.7 vs 15.3%), and AX decreased it (7.2%). For the AJD of NDF, AX performed intermediately in P1; in P2, relative to HF, XY, and AX increased the AJD of NDF (8.4%, 13.1%, and 11.7%, respectively), and in P3, XY, and LF did not differ (13.6 vs. 14.4%). A similar response was observed for the AJD of IDF and TDF, except for XY having the greatest AJD of IDF, T-AX, T-NSP, and TDF in P3 (P < 0.05). Compared to LF, irrespective of period, HF decreased the apparent ileal digestibility (AID) and apparent total tract digestibility (ATTD) of IDF, TDF, and NDF (P < 0.05). Relative to HF, XY partially mitigated this effect, improving the AID and ATTD of TDF, IDF, and NDF (P < 0.05). Increased corn-based fiber decreased nutrient digestibility, but XY partially mitigated that effect in the small intestine through enhanced fiber digestibility when given sufficient adaptation time.

This study investigated the effects of xylanase and arabinoxylan-oligosaccharide supplementation on viscosity, nutrient and energy digestibility in growing pigs fed a high-fiber diet mainly composed of corn over three time periods. Twenty pigs were surgically fitted with cannula in their jejunum and ileum for sample collection. The pigs were randomly assigned to one of four dietary treatment groups: low-fiber control, high-fiber control, xylanase supplementation, and arabinoxylan-oligosaccharide supplementation. The results showed no significant differences in digesta viscosity among dietary treatments. However, there was an interaction between dietary treatment and time for the digestibility of dry matter, gross energy, and dietary fibers. Initially, xylanase did not impact digestibility, but it improved over time. Conversely, arabinoxylan-oligosaccharide initially improved digestibility but declined by the third period. Findings suggest that the efficacy of xylanase in enhancing nutrient and energy digestibility in pigs fed high-fiber diets may depend on the length of the adaptation period. Xylanase supplementation also demonstrated the potential to counteract the negative effects of high-fiber diets. Further research is needed to optimize the application of xylanase in swine production and determine the optimal conditions for its effectiveness.

Dietary xylo-oligosaccharides and arabinoxylans improved growth efficiency by reducing gut epithelial cell turnover in broiler chickens.

BACKGROUND: One of the main roles of the intestinal mucosa is to protect against environmental hazards. Supplementation of xylo-oligosaccharides (XOS) is known to selectively stimulate the growth of beneficial intestinal bacteria and improve gut health and function in chickens. XOS may have an impact on the integrity of the intestinal epithelia where cell turnover is critical to maintain the compatibility between the digestive and barrier functions. The aim of the study was to evaluate the effect of XOS and an arabinoxylan-rich fraction (AXRF) supplementation on gut function and epithelial integrity in broiler chickens. METHODS: A total of 128 broiler chickens (Ross 308) were assigned into one of two different dietary treatments for a period of 42 d: 1) control diet consisting of a corn/soybean meal-based diet; or 2) a control diet supplemented with 0.5% XOS and 1% AXRF. Each treatment was randomly distributed across 8 pens (n = 8) with 8 chickens each. Feed intake and body weight were recorded weekly. On d 42, one male chicken per pen was selected based on average weight and euthanized, jejunum samples were collected for proteomics analysis. RESULTS: Dietary XOS/AXRF supplementation improved feed efficiency (P < 0.05) from d 1 to 42 compared to the control group. Proteomic analysis was used to understand the mechanism of improved efficiency uncovering 346 differentially abundant proteins (DAP) (Padj < 0.00001) in supplemented chickens compared to the non-supplemented group. In the jejunum, the DAP translated into decreased ATP production indicating lower energy expenditure by the tissue (e.g., inhibition of glycolysis and tricarboxylic acid cycle pathways). In addition, DAP were associated with decreased epithelial cell differentiation, and migration by reducing the actin polymerization pathway. Putting the two main pathways together, XOS/AXRF supplementation may decrease around 19% the energy required for the maintenance of the gastrointestinal tract. CONCLUSIONS: Dietary XOS/AXRF supplementation improved growth efficiency by reducing epithelial cell migration and differentiation (hence, turnover), actin polymerization, and consequently energy requirement for maintenance of the jejunum of broiler chickens.

Using in feed xylanase or stimbiotic to reduce the variability in corn nutritive value for broiler chickens.

This study investigated the effects of xylanase and stimbiotic (fiber fermentation enhancer) on the response of broiler chickens fed different corn varieties and determine correlations between variables of interest. Four corn genetic varieties were selected based on their range in nutrient composition. Diets containing 600 g/kg of each corn were supplemented with 0 or 100 g/ton of xylanase or stimbiotic. A total of 1,152 one-day-old male broiler chicks (Ross 308) were divided into 12 treatments, each with 8 pens and 12 birds per pen, for a 21-day study. On d 21, performance parameters were measured, and the ileal energy and organic matter (OM) digestibility and cecal xylanase activity determined. Stimbiotic supplementation improved mFCR compared with all other treatments. There was a treatment by corn variety interaction for body weight (BW), BW gain and coefficient of variation (CV) of BW (P ≤ 0.05). Birds fed corn Variety 1 (highest neutral dietary fiber, protein and soluble arabinoxylan content) supplemented with stimbiotic had the highest BW, while Variety 2 control diet had the lowest. The BW CV in corn Variety 2 was the highest, which improved with stimbiotic supplementation. The BW CV in corn Variety 1 responded better to stimbiotic than xylanase. There were no treatment differences on BW CV in corn Varieties 3 and 4. The lowest OM digestibility was observed in birds fed corn Variety 1 with xylanase, and the highest value was associated with corn Variety 3 with xylanase (highest total arabinoxylan). Xylanase and stimbiotic supplementation increased the endogenous xylanase activity regardless of the corn variety (P ≤ 0.05). Positive correlations between corn fiber contents and phytic acid and the arabinose:xylose ratio were seen, while nonstarch polysaccharide content was negatively correlated with apparent metabolizable energy. In conclusion, corn variety influenced nutrient digestibility and broiler chicken growth. The response to supplementation with xylanase or stimbiotic varied based on the nutritional profile of corn with regards to improvements in digestibility and performance in broiler chickens.

Endogenous mucin conveyed to the mucosa with microbes can assure lumen fermentation and large intestinal security-swine versus fowl.

Endogenous protein leaving the ileum largely consists of accrued mucins from the upper gastrointestinal tract (GIT) that had resisted digestion. The amounts released rely on their mucosal generation during enteral feeding which vary with age as well as diet. These digestion resistant proteins of endogenous origin continue to be unavailable in the large intestine, whereas those of dietary origin provide amino acids that largely support the existing microbial population while denying limited amounts for absorption. Other mucins pre-exist within the large intestine as two layers at the lumen surface. A loose layer harboring a diverse microbial population is superimposed on the unstirred water layer (USWL) which simultaneously acts as an obstacle to microbes at the loose layer while performing as a molecular sieve for nutrients. The USWL is formed through interplay between enterocyte and goblet cells; however, the basis for presence of the loose layer is elusive. Large intestinal fermentation predominates within the colon of swine, whereas fowl employ their ceca. Motility within the colon of swine segregates fine materials into haustrae out-pocketings that parallel their placement within the ceca of fowl. Viscous mucins from small intestinal endogenous losses may envelop microbes within the large intestinal lumen to present successive adherents on the USWL that assemble its loose layer. The loose layer continually functions as a microbial reservoir in support of lumen fermentation. Microbial catabolism of mucin within the loose layer is known to be slow, but its proximity to the enterocyte is of advantage to enterocyte absorption with by-product amino acids fostering the USWL.