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CRISPR-Cas systems can be utilized as programmable-spectrum antimicrobials to combat bacterial infections. However, how CRISPR nucleases perform as antimicrobials across target sites and strains remains poorly explored. Here, we address this knowledge gap by systematically interrogating the use of CRISPR antimicrobials using multidrug-resistant and hypervirulent strains of Klebsiella pneumoniae as models. Comparing different Cas nucleases, DNA-targeting nucleases outperformed RNA-targeting nucleases based on the tested targets. Focusing on AsCas12a that exhibited robust targeting across different strains, we found that the elucidated modes of escape varied widely, restraining opportunities to enhance killing. We also encountered individual guide RNAs yielding different extents of clearance across strains, which were linked to an interplay between improper gRNA folding and strain-specific DNA repair and survival. To explore features that could improve targeting across strains, we performed a genome-wide screen in different K. pneumoniae strains that yielded guide design rules and trained an algorithm for predicting guide efficiency. Finally, we showed that Cas12a antimicrobials can be exploited to eliminate K. pneumoniae when encoded in phagemids delivered by T7-like phages. Altogether, our results highlight the importance of evaluating antimicrobial activity of CRISPR antimicrobials across relevant strains and define critical parameters for efficient CRISPR-based targeting.
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Sistemas CRISPR-Cas , Klebsiella pneumoniae , ARN Guía de Sistemas CRISPR-Cas , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/efectos de los fármacos , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Infecciones por Klebsiella/tratamiento farmacológico , Infecciones por Klebsiella/microbiología , Proteínas Asociadas a CRISPR/metabolismo , Proteínas Asociadas a CRISPR/genética , Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Genoma Bacteriano/genética , Edición Génica/métodos , HumanosRESUMEN
Studies on the bioavailability, serum levels, and absorption of hydrolyzable tannin compounds are lacking. In this study, we performed a pharmacokinetic trial, measured the serum levels of compounds in broilers that were reared with different feed added or not with tannins, and tested the digestibility of tannins throughout the intestinal tract. Only gallic acid and 4-O-methyl gallic acid were found in the serum. Moreover, gallic acid showed a 41.8% absolute oral bioavailability and a 72.3% relative bioavailability of gallic acid from chestnut extract compared to the standard. The rapid metabolization caused alternating serum levels during the day and night. These patterns were not affected by the feed type or the previous addition of tannins in the feed. The absorption and metabolization in the intestines occurred gradually throughout the intestinal tract. The latter was true for gallic acid as well as ellagic acid, which was not found in the serum. We can conclude that components from chestnut tannins are absorbed throughout all components of the intestinal tract and are eliminated quickly with little interaction from the feed and previous addition of tannins. Moreover, ellagic acid seems to be absorbed but would remain accumulated in the intestinal tissue or be metabolized by the microbiome.
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Ácido Gálico , Taninos , Animales , Ácido Elágico , Pollos , Madera , DietaRESUMEN
Klebsiella pneumoniae (Kp), a human gut colonizer and opportunistic pathogen, is a major contributor to the global burden of antimicrobial resistance. Virulent bacteriophages represent promising agents for decolonization and therapy. However, the majority of anti-Kp phages that have been isolated thus far are highly specific to unique capsular types (anti-K phages), which is a major limitation to phage therapy prospects due to the highly polymorphic capsule of Kp. Here, we report on an original anti-Kp phage isolation strategy, using capsule-deficient Kp mutants as hosts (anti-Kd phages). We show that anti-Kd phages have a broad host range, as the majority are able to infect noncapsulated mutants of multiple genetic sublineages and O-types. Additionally, anti-Kd phages induce a lower rate of resistance emergence in vitro and provide increased killing efficiency when in combination with anti-K phages. In vivo, anti-Kd phages are able to replicate in mouse guts colonized with a capsulated Kp strain, suggesting the presence of noncapsulated Kp subpopulations. The original strategy proposed here represents a promising avenue that circumvents the Kp capsule host restriction barrier, offering promise for therapeutic development. IMPORTANCE Klebsiella pneumoniae (Kp) is an ecologically generalist bacterium as well as an opportunistic pathogen that is responsible for hospital-acquired infections and a major contributor to the global burden of antimicrobial resistance. In the last decades, limited advances have been made in the use of virulent phages as alternatives or complements to antibiotics that are used to treat Kp infections. This work demonstrates the potential value of an anti-Klebsiella phage isolation strategy that addresses the issue of the narrow host range of anti-K phages. Anti-Kd phages may be active in infection sites in which capsule expression is intermittent or repressed or in combination with anti-K phages, which often induce the loss of capsule in escape mutants.
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Bacteriófagos , Infecciones por Klebsiella , Animales , Humanos , Ratones , Klebsiella pneumoniae/genética , Bacteriófagos/genética , Especificidad del Huésped , Antibacterianos/farmacología , Klebsiella , Infecciones por Klebsiella/terapia , Infecciones por Klebsiella/microbiologíaRESUMEN
Horizontal gene transfer is an important evolutionary force, facilitating bacterial diversity. It is thought to be pervasive in host-associated microbiomes, where bacterial densities are high and mobile elements are frequent. These genetic exchanges are also key for the rapid dissemination of antibiotic resistance. Here, we review recent studies that have greatly extended our knowledge of the mechanisms underlying horizontal gene transfer, the ecological complexities of a network of interactions involving bacteria and their mobile elements, and the effect of host physiology on the rates of genetic exchanges. Furthermore, we discuss other, fundamental challenges in detecting and quantifying genetic exchanges in vivo, and how studies have contributed to start overcoming these challenges. We highlight the importance of integrating novel computational approaches and theoretical models with experimental methods where multiple strains and transfer elements are studied, both in vivo and in controlled conditions that mimic the intricacies of host-associated environments.
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Transferencia de Gen Horizontal , Microbiota , Bacterias/genética , Evolución Biológica , Microbiota/genética , Antibacterianos/farmacologíaRESUMEN
It is known that high doses of various tannins could impair broiler growth, and this seems to be linked to a lowered protein availability. However, effects on protein digestion under the influence of hydrolysable tannins were minimal in previous research and literature. Other possible proposed reasons to explain reduced growth are scarce. In this experiment we studied the effect of hydrolysable tannins on body allometry by using different feeding schemes throughout the rearing period. In total 112 individually reared male Ross 308 broilers received a 3-phase basal diet with chestnut wood extract (+: 2,000 mg/kg) or not (-: 0 mg/kg) (Tanno-SAN®, Sanluc International NV, Belgium). This resulted in 2 groups during the starter period (S+, S-), 4 groups in the grower period (G++, G+-, G-+, G-) and 8 groups in the finisher period (F+++, F++-, F+-+, F+--, F-++, F-+-, F--+, F--). Similar to previous studies, growth reduction was also observed in this study. Effects were the largest in broilers that were given the tannins during the grower phase. At the end of each phase 8 broilers per group were euthanized and sampled. Liver, pancreas, pectoralis muscle, intestinal weights and intestinal length were recorded. The largest effects were seen on the intestine. Broilers that received tannins during the grower phase, had longer intestines at the end of the finisher period. Furthermore, histological differences between treatment groups were observed at the end of the grower period. Addition of tannins in the grower phase (G-+, G++) resulted in longer villi, whereas addition of tannins in the starter (G+-, G++) caused deeper crypts at the end of the grower phase, with the group (G-+) having the highest villi-to-crypt ratio. These results tentatively prove that tannins influence intestinal growth, both macroscopically as well as histologically. We hypothesize that the observed growth reduction with tannins could be the result of a changed energy and nutrient partitioning, i.e., more nutrients are directed to intestinal growth than for muscle growth.
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Abundance and diversity of bacteria and their viral predators, bacteriophages (phages), in the digestive tract are associated with human health. Particularly intriguing is the long-term coexistence of these two antagonistic populations. We performed genome-wide RNA sequencing on a human enteroaggregative Escherichia coli isolate to identify genes differentially expressed between in vitro conditions and in murine intestines. We experimentally demonstrated that four of these differentially expressed genes modified the interactions between E. coli and three virulent phages by either increasing or decreasing its susceptibility/resistance pattern and also by interfering with biofilm formation. Therefore, the regulation of bacterial genes expression during the colonization of the digestive tract influences the coexistence of phages and bacteria, highlighting the intricacy of tripartite relationships between phages, bacteria, and the animal host in intestinal homeostasis.
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Bacteriófagos , Animales , Bacterias/genética , Bacteriófagos/fisiología , Escherichia coli/genética , Expresión Génica , Genes Bacterianos , RatonesRESUMEN
In 2 broiler trials, the effects of chestnut tannins on performance and meat quality (trial 1), and digestion (trial 2) were evaluated. In both trials, Ross 308 broilers received one of 2 basal diets: one basal diet contained corn and soy as main feed ingredients, while the challenge basal diet contained wheat, palm oil, and rapeseed meal. The composition of the basal diets was chosen to assess the interaction between chestnut tannins and diet composition. To both basal diets, chestnut tannins were added at 3 doses: 0 mg/kg (T-), 500 mg/kg (T+), or 2,000 mg/kg (T++), resulting in a total of 6 treatments. In trial 1, both basal diets containing 2,000 mg/kg chestnut tannins lowered broiler performance in grower and finisher phases. A tannin dose of 500 mg/kg had no effect on performance in either basal diet. Corn-based diets resulted in lower meat pH compared to wheat diets. Further, addition of chestnut tannins resulted in increased meat pH, and caused proportionally a lower meat drip loss and shear force for both basal diets. During the digestibility study (trial 2), blood was also collected. None of the treatments affected digestibility or blood parameters (glucose, non-esterified fatty acids, and triacylglycerols). Malondialdehyde (MDA) was measured in plasma to assess antioxidative properties of chestnut tannins. In wheat diets, chestnut tannins significantly lowered plasma MDA demonstrating its antioxidative nature. Regarding gut health, crypt depth decreased proportionally with the dosage of chestnut tannins in both basal diets with significantly shallower crypts for the wheat diets compared to the corn diets. Relative intestinal growth was stimulated in the wheat diets proportionally to the tannin dose based on the larger relative gut length. In conclusion, chestnut tannins did not influence digestive metabolism, yet they lowered performance at higher doses regardless of feed ingredients used in the diet. Tannins positively affected meat quality and when added to wheat diets, intestinal growth was stimulated and the antioxidative status of the broilers improved.
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Pollos , Taninos , Alimentación Animal/análisis , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Dieta/veterinaria , Suplementos Dietéticos , Digestión , Carne/análisis , NutrientesRESUMEN
Forecasting COVID-19 disease severity is key to supporting clinical decision making and assisting resource allocation, particularly in intensive care units (ICUs). Here, we investigated the utility of time- and frequency-related features of the backscattered signal of serum patient samples to predict COVID-19 disease severity immediately after diagnosis. ICU admission was the primary outcome used to define disease severity. We developed a stacking ensemble machine learning model including the backscattered signal features (optical fingerprint), patient comorbidities, and age (AUROC = 0.80), which significantly outperformed the predictive value of clinical and laboratory variables available at hospital admission (AUROC = 0.71). The information derived from patient optical fingerprints was not strongly correlated with any clinical/laboratory variable, suggesting that optical fingerprinting brings unique information for COVID-19 severity risk assessment. Optical fingerprinting is a label-free, real-time, and low-cost technology that can be easily integrated as a front-line tool to facilitate the triage and clinical management of COVID-19 patients.
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Nontyphoidal Salmonella bacteria are the causative agent of salmonellosis, which accounts for the majority of foodborne illness of bacterial etiology in humans. Here, we demonstrate the safety and efficacy of the prophylactic administration of a bacteriophage preparation termed FOP (foodborne outbreak pill), which contains lytic phages targeting Salmonella (SalmoFresh phage cocktail), Shiga toxin-producing Escherichia coli (STEC), and Listeria monocytogenes, for lowering Salmonella burdens in OMM12 gnotobiotic mice. Prophylactic administration of FOP significantly reduced the levels of Salmonella in feces and in intestinal sections compared to the levels in controls. Moreover, the overall symptoms of the disease were also considerably lessened. Dose-dependent administration of FOP showed that phage amplification reached similarly high levels in less than 48 h independent of dose. In addition, 16S rRNA gene analysis showed that FOP did not alter the intestinal microbiota of healthy OMM12 mice and reduced microbiota perturbations induced by Salmonella. FOP maintained its full potency against Salmonella in comparison to that of SalmoFresh, its Salmonella-targeting component phages alone. Altogether, the data support that preventive administration of FOP may offer a safe and effective approach for reducing the risk of foodborne infections caused by Salmonella and, potentially, other foodborne bacteria (namely, STEC and L. monocytogenes) targeted by the FOP preparation. IMPORTANCE Foodborne bacterial infections cause worldwide economic loss. During an epidemic, the use of antibiotics to slow down the spread of the disease is not recommended because of their side effects on the resident microbiota and the selection of antibiotic-resistant bacteria. Here, we investigated the potential for the prophylactic administration of bacteriophages (viruses infecting bacteria) to reduce the burden of Salmonella in vivo using mice colonized by a synthetic microbiota. We found that the repeated administration of bacteriophages was safe and efficient in lowering the Salmonella burden. Perturbations of the microbiota by the Salmonella infection were also reduced when mice received bacteriophages. Altogether, these data support the use of bacteriophages as a prophylactic intervention to lower the spread of foodborne epidemics.
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Terapia de Fagos , Infecciones por Salmonella/prevención & control , Salmonella typhimurium/virología , Animales , Heces/microbiología , Femenino , Microbioma Gastrointestinal , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Profilaxis Pre-Exposición , Infecciones por Salmonella/microbiología , Salmonella typhimurium/fisiologíaRESUMEN
The dry season in tropical regions urges livestock to feed on nutritionally very poor diets. It has not been explored how tropical sheep-assumed grazers-and tropical goats-intermediate browsers-cope with a high-fibre tannin-rich diet. This study was designed to determine the effects of a high-fibre and tannin-rich diet on the flow of important microminerals iron (Fe), zinc (Zn) and copper (Cu) throughout the digestive tract of tropical sheep and goats. The feeding trial was set up with twelve adult male animals, six sheep with mean body weight (BW) of 30.3 ± 1.6 kg and six goats with mean BW of 26.4 ± 2.2 kg. The feed consisted of 36% leaves of Millettia ferruginea, 61% hay and 3% concentrate and was offered at 3% of BW (all on dry matter (DM) basis). The total faecal collection was carried out for 7 consecutive days. At the end of the experimental period, the animals were slaughtered to collect liver and digesta samples from the gastrointestinal tract. Feed, digesta and faecal samples underwent analysis of Fe, Zn, and Cu and total tannins (TT). Goats had significantly higher reticulum Cu concentrations expressed on DM as compared to sheep. Faecal Cu concentrations were higher for goats compared to sheep. Reticulum and colon digesta Zn levels were higher in goats than sheep. Abomasum and colon Fe levels were higher in sheep than goats when expressed on DM. These results suggest differences in feed intake, micromineral absorption, secretion and excretion between sheep and goats, pointing to a divergent mineral metabolism as an adaptation to the challenge of a dry season diet having very low nutritive value.
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Cabras , Taninos , Alimentación Animal/análisis , Animales , Cobre , Dieta/veterinaria , Digestión , Tracto Gastrointestinal , Hierro , Masculino , Ovinos , ZincRESUMEN
The ecological dynamics underlying the coexistence between antagonistic populations of bacteria and their viruses, bacteriophages (phages), in the mammalian gut microbiota remain poorly understood. We challenged a murine synthetic bacterial community with phages to study the factors allowing phages-bacteria coexistence. Coexistence was not dependent on the development of phage-resistant clones nor on the ability of phages to extend their host range. Instead, our data suggest that phage-inaccessible sites in the mucosa serve as a spatial refuge for bacteria. From there, bacteria disseminate in the gut lumen where they are predated by luminal phages fostering the presence of intestinal phage populations. The heterogeneous biogeography of microbes contributes to the long-term coexistence of phages with phage-susceptible bacteria. This observation could explain the persistence of intestinal phages in humans as well as the low efficiency of oral phage therapy against enteric pathogens in animal models and clinical trials.
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Bacterias/crecimiento & desarrollo , Bacterias/virología , Bacteriófagos/fisiología , Escherichia coli/crecimiento & desarrollo , Escherichia coli/virología , Tracto Gastrointestinal/microbiología , Membrana Mucosa/microbiología , Animales , Ecosistema , Heces/microbiología , Femenino , Microbioma Gastrointestinal , Vida Libre de Gérmenes , Masculino , Ratones , Ratones Endogámicos C57BL , Interacciones Microbianas , Modelos AnimalesRESUMEN
RATIONALE: Pathological biomechanical signaling induces vascular anomalies including cerebral cavernous malformations (CCM), which are caused by a clonal loss of CCM1/KRIT1 (Krev interaction trapped protein 1), CCM2/MGC4607, or CCM3/PDCD10. Why patients typically experience lesions only in lowly perfused venous capillaries of the cerebrovasculature is completely unknown. OBJECTIVE: In contrast, animal models with a complete loss of CCM proteins lack a functional heart and blood flow and exhibit vascular anomalies within major blood vessels as well. This finding raises the possibility that hemodynamics may play a role in the context of this vascular pathology. METHODS AND RESULTS: Here, we used a genetic approach to restore cardiac function and blood flow in a zebrafish model of CCM1. We find that blood flow prevents cardiovascular anomalies including a hyperplastic expansion within a large Ccm1-deficient vascular bed, the lateral dorsal aorta. CONCLUSIONS: This study identifies blood flow as an important physiological factor that is protective in the cause of this devastating vascular pathology.
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Velocidad del Flujo Sanguíneo/fisiología , Neoplasias del Sistema Nervioso Central/diagnóstico por imagen , Modelos Animales de Enfermedad , Hemangioma Cavernoso del Sistema Nervioso Central/diagnóstico por imagen , Animales , Animales Modificados Genéticamente , Neoplasias del Sistema Nervioso Central/fisiopatología , Angiografía Cerebral/métodos , Hemangioma Cavernoso del Sistema Nervioso Central/fisiopatología , Pez CebraRESUMEN
The intestinal microbiota is intimately linked to human health. Decoding the mechanisms underlying its stability in healthy subjects should uncover causes of microbiota-associated diseases and pave the way for treatment. Bacteria and bacteriophages (phages) are the most abundant biological entities in the gastrointestinal tract, where their coexistence is dynamic and affixed. Phages drive and maintain bacterial diversity by perpetuating the coevolutionary interactions with their microbial prey. This review brings together recent in silico, in vitro, and in vivo work dissecting the complexity of phage-bacteria interactions in the intestinal microbiota, including coevolution perspectives. We define the types of dynamics encountered in the gastrointestinal tract and the parameters that affect their outcome. The impact of intestinal physiology on phage-bacterial coevolution is analyzed in the light of its potential contribution to the relationship between the microbiota and human health.
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Bacterias/virología , Bacteriófagos/fisiología , Tracto Gastrointestinal/microbiología , Interacciones Microbianas , Animales , Coevolución Biológica , Microbioma Gastrointestinal/fisiología , Humanos , Ratones , Organismos Libres de Patógenos EspecíficosRESUMEN
Viruses that infect bacteria, or bacteriophages, are among the most abundant entities in the gut microbiome. However, their role and the mechanisms by which they infect bacteria in the intestinal tract remain poorly understood. We recently reported that intestinal bacteria are an evolutionary force, driving the expansion of the bacteriophage host range by boosting the genetic variability of these viruses. Here, we expand these observations by studying antagonistic bacteriophage-bacteria coevolution dynamics and revealing that bacterial genetic variability is also increased under the pressure of bacteriophage predation. We propose a model showing how the expansion of bacteriophage-bacteria infection networks is relative to the opportunities for coevolution encountered in the intestinal tract. Our data suggest that predator-prey dynamics are perpetuated and differentiated in parallel, to generate and maintain intestinal microbial diversity and equilibrium.
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Bacterias/virología , Bacteriófagos/fisiología , Coevolución Biológica , Microbioma Gastrointestinal/fisiología , Adaptación Fisiológica , Animales , Antibiosis/genética , Bacterias/genética , Bacteriófagos/genética , Biodiversidad , Microbioma Gastrointestinal/genética , Variación Genética , Humanos , Intestinos/microbiología , Intestinos/virología , Modelos BiológicosRESUMEN
Phage therapy is based on a simple concept: the use of a virus (bacteriophage) that is capable of killing specific pathogenic bacteria to treat bacterial infections. Since the pioneering work of Félix d’Herelle, bacteriophages (phages) isolated in vitro have been shown to be of therapeutic value. Over decades of study, a large number of rather complex mechanisms that are used by phages to hijack bacterial resources and to produce their progeny have been deciphered. While these mechanisms have been identified and have been studied under optimal conditions in vitro, much less is known about the requirements for successful viral infections in relevant natural conditions. This is particularly true in the context of phage therapy. Here, we highlight the parameters affecting phage replication in both in vitro and in vivo environments, focusing, in particular, on the mammalian digestive tract. We propose avenues for increasing the knowledge-guided implementation of phages as therapeutic tools.
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Bacterias/virología , Bacteriófagos/crecimiento & desarrollo , Interacciones Huésped-Parásitos , Animales , Tracto Gastrointestinal/microbiología , Tracto Gastrointestinal/virología , Humanos , MamíferosRESUMEN
Viscosity of intestinal contents is known to affect digestion and absorption of nutrients. In most poultry studies, intestinal viscosity has been measured only after complete removal of solid particles by centrifugation. Centrifugation may however remove particles that contribute to viscosity, hence giving rise to an underestimation of viscosity. Two viscosity measurement techniques, one including a centrifugation step (Brookfield) and the other without (Haake), were compared in-vitro to assess whether both techniques result in similar conclusions regarding viscosity in feedstuffs. Two sets of feedstuff preparations were used. The first set was prepared with different combinations of milled feedstuffs in order to have a wide range of viscosity: 100% corn, 25% corn + 75% wheat, 100% wheat, 90% wheat + 10% rye, all mixed with distilled water. In the second set, barley was incubated with different beta-glucanases, and soybean and sunflower meal were incubated with different pectinases, again all mixed with distilled water. Viscosity was assessed using both techniques (Haake and Brookfield equipments) at six different time points. To evaluate the extent of agreement between the two methods, the Lin's concordance correlation coefficient (CCC) was assessed using the percentage of increase in viscosity within each method, based on pairwise feedstuffs comparison (first set), or relative to the feedstuff without enzyme (second set). The rate of the agreement between the two methods was substantial for the first set of feedstuffs (66%) and for the barley diets incubated with beta-glucanases (69%), whereas the CCC score for the soybean meal diets was very poor (2%) and fair for the sunflower meal diets, incubated with pectinases (32%). The lack of agreement for the latter can be explained by the limited variation in viscosity in these low-viscous mixtures. Although the two techniques are considerably different (e.g., with or without preceding particle removal), they seem to render similar conclusions when applied to poultry feedstuffs to identify distinct differences under the tested circumstances.
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Alimentación Animal/análisis , Crianza de Animales Domésticos/métodos , Contenido Digestivo/química , Crianza de Animales Domésticos/instrumentación , Animales , Centrifugación , Aves de Corral , ViscosidadRESUMEN
Endothelial cells respond to different levels of fluid shear stress through adaptations of their mechanosensitivity. Currently, we lack a good understanding of how this contributes to sculpting of the cardiovascular system. Cerebral cavernous malformation (CCM) is an inherited vascular disease that occurs when a second somatic mutation causes a loss of CCM1/KRIT1, CCM2, or CCM3 proteins. Here, we demonstrate that zebrafish Krit1 regulates the formation of cardiac valves. Expression of heg1, which encodes a binding partner of Krit1, is positively regulated by blood-flow. In turn, Heg1 stabilizes levels of Krit1 protein, and both Heg1 and Krit1 dampen expression levels of klf2a, a major mechanosensitive gene. Conversely, loss of Krit1 results in increased expression of klf2a and notch1b throughout the endocardium and prevents cardiac valve leaflet formation. Hence, the correct balance of blood-flow-dependent induction and Krit1 protein-mediated repression of klf2a and notch1b ultimately shapes cardiac valve leaflet morphology.
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Células Endoteliales/fisiología , Válvulas Cardíacas/embriología , Factores de Transcripción de Tipo Kruppel/metabolismo , Mecanotransducción Celular , Glicoproteínas de Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Proteínas MuscularesRESUMEN
BACKGROUND: High protein diets shift the faecal microbiota into a more unfavourable composition in obese humans. In lean dogs, higher protein consumption is accompanied with increased production of putrefactive fermentation products, whereas obese dogs have a different gut microbiota compared to lean dogs. Still, the impact of high dietary protein on gut microbiota in obese dogs remains unclear. The aim of this study was to investigate faecal microbial changes in lean and obese dogs in response to two different levels of dietary protein. Six healthy lean and six obese Beagles were fed a high protein diet (HP) and a low protein diet (LP) for 28 days each in a crossover design. Denaturing gradient gel electrophoresis and quantitative PCR were performed on faecal samples for microbial profiling. Plasma acylcarnitine and fermentation metabolites were measured. RESULTS: Dogs fed HP had higher concentrations of protein fermentation metabolites including faecal ammonia, isovalerate, isobutyrate, phenol, indole, serum indoxyl sulphate and plasma 3-OH isovalerylcarnitine compared to dogs fed LP, whereas no changes in faecal concentrations of acetate and butyrate were observed. The abundances of clostridial clusters IV and XIVa, covering the majority of butyrate-producing bacteria, and of the butyrate kinase gene, one of the terminal genes of the butyrate synthesis pathway were higher in dogs on HP compared to LP. Significant interactions between diet and body condition were found for the abundance of Firmicutes, Lactobacillus and clostridial cluster I. The similarity coefficient of faecal microbiota between the two diets was smaller in obese dogs than in lean dogs. CONCLUSIONS: High protein diet increased the abundance and activity of butyrate-producing bacteria in Beagles independent of the body condition. In addition, increasing dietary protein content had a greater overall impact on faecal microbiota in obese compared to lean dogs.
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Proteínas en la Dieta/farmacología , Enfermedades de los Perros/microbiología , Perros/metabolismo , Heces/microbiología , Microbioma Gastrointestinal/fisiología , Obesidad/veterinaria , Animales , Estudios de Casos y Controles , ADN Bacteriano/genética , Enfermedades de los Perros/metabolismo , Enfermedades de los Perros/fisiopatología , Perros/fisiología , Femenino , Microbioma Gastrointestinal/genética , Masculino , Obesidad/metabolismo , Obesidad/microbiología , Obesidad/fisiopatología , Reacción en Cadena de la Polimerasa/veterinariaRESUMEN
The evolution of new strains within the gut ecosystem is poorly understood. We used a natural but controlled system to follow the emergence of intraspecies diversity of commensal Escherichia coli, during three rounds of adaptation to the mouse gut (â¼1,300 generations). We previously showed that, in the first round, a strongly beneficial phenotype (loss-of-function for galactitol consumption; gat-negative) spread to >90% frequency in all colonized mice. Here, we show that this loss-of-function is repeatedly reversed when a gat-negative clone colonizes new mice. The regain of function occurs via compensatory mutation and reversion, the latter leaving no trace of past adaptation. We further show that loss-of-function adaptive mutants reevolve, after colonization with an evolved gat-positive clone. Thus, even under strong bottlenecks a regime of strong-mutation-strong-selection dominates adaptation. Coupling experiments and modeling, we establish that reverse evolution recurrently generates two coexisting phenotypes within the microbiota that can or not consume galactitol (gat-positive and gat-negative, respectively). Although the abundance of the dominant strain, the gat-negative, depends on the microbiota composition, gat-positive abundance is independent of the microbiota composition and can be precisely manipulated by supplementing the diet with galactitol. These results show that a specific diet is able to change the abundance of specific strains. Importantly, we find polymorphism for these phenotypes in indigenous Enterobacteria of mice and man. Our results demonstrate that natural selection can greatly overwhelm genetic drift at structuring the strain diversity of gut commensals and that competition for limiting resources may be a key mechanism for maintaining polymorphism in the gut.