Short-term physiological responses to moderate heat stress in grazing dairy cows in temperate climate.

Even in temperate climate regions, an increase in ambient temperature and exposure to solar radiation can cause heat stress in lactating dairy cows. We hypothesised that grazing dairy cows exhibit short-term physiological changes due to increasing heat load under moderate climate conditions. Over two consecutive summers, 38 lactating Holstein dairy cows were studied in a full-time grazing system. Data were collected in 10 experimental periods of up to three consecutive days with a moderate comprehensive climate index (CCI). The individual animals' vaginal temperature (VT), heart rate, and locomotor activity data were automatically monitored with sensors. Blood samples and proportional whole milk samples were collected at afternoon milking. The concentrations of beta-hydroxybutyrate, glucose, non-esterified fatty acids, urea nitrogen, plasma thyroxine and triiodothyronine were analysed in blood plasma, and fat, protein, lactose, urea nitrogen, cortisol, Na+, K+, and Cl- concentrations were analysed in milk. The daily distribution of VT recordings greater than 39 °C showed a circadian rhythm with a proportion of recordings of 2% and lower during the night and a percentage of 10% or higher in the afternoon. The cows' maximal daily vaginal temperature (VTMAX) between 0830 and 1430 h was positively related to the mean daily CCI in the same time period (CCIMEAN; mean and SD 23.6 ± 5.4 °C). Cows with greater VTMAX had an increased mean heart rate, plasma glucose and milk cortisol concentrations and decreased concentrations of plasma thyroxine and triiodothyronine. The concentration of Na+ in milk was lower, and the concentration of K+ in milk tended to be higher in cows with increased VTMAX. For beta-hydroxybutyrate, non-esterified fatty acids and urea nitrogen concentrations in plasma and fat and lactose concentrations in milk no relationships were found in terms of increasing VT. For milk urea nitrogen and protein concentrations, the proportion of total variance explained by inter-individual or -period variance was high. In conclusion, changes observed in milk and blood likely reflected short-term physiological responses to moderate heat stress. In particular, milk cortisol and Na+ may be useful traits for timely monitoring of heat stress in individual cows because their inter-individual variances were relatively small and samples can be collected non-invasively.

Dark Side of Ammonium Nitrogen in Paddy Soil with Low Organic Matter: Stimulation of Microbial As(V) Reduction and As(III) Transfer from Soil to Rice Grains.

The bioavailability of arsenic (As) is influenced by ammonium (NH4+-N) fertilization, but the underlying mechanisms controlling As transformation in soil-rice systems are still not fully understood. The effects of two NH4+-N fertilizers, urea and NH4HCO3, on the transformation of As in a paddy soil with low organic matter content and transfer in rice plants were investigated. Treatments with urea and NH4HCO3 significantly increased arsenite (As(III)) concentration in porewater, bioavailable As in rhizosphere soil, and the relative abundance of the As(V) respiratory reductase gene (arrA) and As(III) methyltransferase gene (arsM). Furthermore, the relative expression of As transporter genes in rice roots, such as OsLsi1, OsLsi2, and OsLsi3, was upregulated, and the translocation efficiency of As(III) from rice roots to brown rice was promoted. Subsequently, As(III) accumulation in brown rice significantly increased. Therefore, attention should be paid to As-contaminated paddy fields with NH4+-N fertilization.

Evaluation of drug carrier hepatotoxicity using primary cell culture models.

This study aims to establish a primary rat hepatocyte culture model to evaluate dose-dependent hepatotoxic effects of drug carriers (lipopolymer nanoparticles; LPNs) temporal. Primary rat hepatocyte cell cultures were used to determine half-maximal Inhibition Concentrations (IC50) of the drug-carrier library. Drug-carrier library, at concentrations <50 µg/mL, is benign to primary rat hepatocytes as determined using albumin and urea secretions. Albumin, as a hepatic biomarker, exhibited a more sensitive and faster outcome, compared to urea, for the determination of the IC50 value of LPNs. Temporal measurements of hepatic biomarkers including urea and albumin, and rigorous physicochemical (hydrodynamic diameter, surface charge, etc.) characterization, should be combined to evaluate the hepatotoxicity of drug carrier libraries in screens.

Feeding lower-protein diets based on red clover and grass or alfalfa and corn silage does not affect milk production but improves nitrogen use efficiency in dairy cows.

Reducing the dietary crude protein (CP) concentration can decrease the financial cost and lower the environmental impact of milk production. Two studies were conducted to examine the effects of reducing the dietary CP concentration on animal performance, nutrient digestibility, milk fatty acid (FA) profile, and nitrogen use efficiency (NUE; milk N/N intake) in dairy cows fed legume silage-based diets. Thirty-six multiparous Holstein-Friesian dairy cows that were 76 ± 14 (mean ± SD) days in milk and 698 ± 54 kg body weight were used in a 3 × 3 Latin square design in each of 2 studies, with 3 periods of 28 d. In study 1, cows were fed diets based on a 50:50 ratio of red clover to grass silage [dry matter (DM) basis] containing 1 of 3 dietary CP concentrations: high (H) = 175 g of CP/kg of DM; medium (M) = 165 g of CP/kg of DM; or low (L) = 150 g of CP/kg of DM. In study 2, cows were fed 175 g of CP/kg of DM with a 50:50 ratio of alfalfa to corn silage (H50) or 1 of 2 diets containing 150 g of CP/kg of DM with either a 50:50 (L50) or a 60:40 (L60) ratio of alfalfa to corn silage. Cows in both studies were fed a total mixed ration with a forage-to-concentrate ratio of 52:48 (DM basis). All diets were formulated to meet the MP requirements, except L (95% of MP requirements). In study 1, cows fed L ate 1.6 kg of DM/d less than those fed H or M, but milk yield was similar across treatments. Mean milk protein, fat, and lactose concentrations were not affected by diet. However, the apparent total-tract nutrient digestibility was decreased in cows fed L. The NUE was 5.7 percentage units higher in cows fed L than H. Feeding L also decreased milk and plasma urea concentrations by 4.4 mg/dL and 0.78 mmol/L, respectively. We found no effect of dietary treatment on the milk saturated or monounsaturated FA proportion, but the proportion of polyunsaturated FA was increased, and milk odd- and branched-chain FA decreased in cows fed L compared with H. In study 2, DM intake was 2 kg/d lower in cows receiving L50 than H50. Increasing the alfalfa content and feeding a low-CP diet (L60) did not alter DMI but decreased milk yield and milk protein concentration by 2 kg/d and 0.6 g/kg, respectively, compared with H50. Likewise, milk protein and lactose yield were decreased by 0.08 kg/d in cows receiving L60 versus H50. Diet had no effect on apparent nutrient digestibility. Feeding the low-CP diets compared with H50 increased the apparent NUE by approximately 5 percentage units and decreased milk and plasma urea concentrations by 7.2 mg/dL and 1.43 mmol/L, respectively. Dietary treatment did not alter milk FA profile except cis-9,trans-11 conjugated linoleic acid, which was higher in milk from cows receiving L60 compared with H50. We concluded that reducing CP concentration to around 150 g/kg of DM in red clover and grass or alfalfa and corn silage-based diets increases the apparent NUE and has little effect on nutrient digestibility or milk performance in dairy cows.

Dietary protein oscillation: effects on digestibility, nutrient balance and estimated microbial protein synthesis in lactating dairy cows.

Various studies with growing ruminants report increases in nitrogen use efficiency (NUE) when feeding oscillating (OS) dietary CP, whereas limited research with lactating dairy cows demonstrates a lack of improvement in NUE when feeding OS diets. We hypothesised that a total mixed ration (TMR) delivering OS CP (48-h phases of 134 and 171 g CP/kg DM, respectively) compared to a static CP TMR (ST; 152 g CP/kg DM) would result in similar or increased urinary purine derivative excretion (as a marker of microbial protein synthesis (MPS)) and greater urinary nitrogen excretion in lactating dairy cows. Responses in intake, production, apparent total tract digestibility (ATTD), nutrient balance, and estimated MPS were evaluated using faecal and urine collection in 12 multiparous cows (172 ± 39 d in milk) in a randomised complete block design, where total urinary output was estimated indirectly. All measurements were taken during d 8 (at 1700) to d 16 (at 1700) of the 16-d study that followed a 28-d period in which cows already received their respective treatments. Dry matter intake, yields of milk, protein, fat, lactose, and fat- and protein-corrected milk were similar for ST and OS. Milk composition, BW, and body condition score also did not differ between treatments, except for a tendency for increased milk urea concentration with OS (13.7 vs 12.4 mg/dL). Feed efficiency, NUE and ATTD of organic matter, NDF, CP and gross energy did not differ, but ATTD of crude fat (658 vs 627 g/kg) and starch (980 vs 975 g/kg) increased, and ATTD of DM (702 vs 691 g/kg) tended to increase with OS. Milk energy as a proportion of digested energy tended to decrease with OS (34.6 vs 37.1%), but other energy metabolism variables were not affected by treatment. Estimated urinary nitrogen excretion increased (165 vs 144 g/d), estimated urinary nitrogen as a proportion of nitrogen intake tended to increase (25.3 vs 22.7%), and milk nitrogen as a proportion of digested nitrogen decreased (47.3 vs 51.8%) in response to OS. Estimated urinary excretion of creatinine (184 vs 165 mmol/d), uric acid (29 vs 20 mmol/d) and urea (3.1 vs 2.5 mol/d) increased, but other nitrogen metabolism parameters were not affected by OS. Overall, oscillating dietary CP content did not affect lactational performance, milk NUE, or estimated MPS. However, ATTD of some nutrients increased, postabsorptive energy use for milk synthesis tended to decrease, and estimated urinary nitrogen losses increased with OS.

Constraining the composition and quantity of organic matter used by abundant marine Thaumarchaeota.

Marine Group I (MGI) Thaumarchaeota were originally described as chemoautotrophic nitrifiers, but molecular and isotopic evidence suggests heterotrophic and/or mixotrophic capabilities. Here, we investigated the quantity and composition of organic matter assimilated by individual, uncultured MGI cells from the Pacific Ocean to constrain their potential for mixotrophy and heterotrophy. We observed that most MGI cells did not assimilate carbon from any organic substrate provided (glucose, pyruvate, oxaloacetate, protein, urea, and amino acids). The minority of MGI cells that did assimilate it did so exclusively from nitrogenous substrates (urea, 15% of MGI and amino acids, 36% of MGI), and only as an auxiliary carbon source (<20% of that subset's total cellular carbon was derived from those substrates). At the population level, MGI assimilation of organic carbon comprised just 0.5%-11% of total biomass carbon. We observed extensive assimilation of inorganic carbon and urea- and amino acid-derived nitrogen (equal to that from ammonium), consistent with metagenomic and metatranscriptomic analyses performed here and previously showing a widespread potential for MGI to perform autotrophy and transport and degrade organic nitrogen. Our results constrain the quantity and composition of organic matter used by MGI and suggest they use it primarily to meet nitrogen demands for anabolism and nitrification.

Live yeast supplementation altered the bacterial community's composition and function in rumen and hindgut and alleviated the detrimental effects of heat stress on dairy cows.

The objective of this study was to investigate the effects of live yeast (LY, Saccharomyces cerevisiae) on the lactation performance, bacterial community, and functions in the rumen and hindgut of dairy cows under heat stress. Thirty-three multiparous (parity 3.9 ± 0.8) Holstein dairy cows (189.1 ± 6.6 d in milk at the beginning of the experiment) were randomly assigned to three groups (11 cows per treatment). Cows in the three groups were fed a diet without yeast (CON), with 10 g yeast/d/head (LY-10), and with 20 g yeast/d/head (LY-20). The yeast product contained 2.0 × 1010 CFU/g. Supplementing LY decreased the rectal temperature and respiratory rate of cows, and increased dry matter intake, milk yield, milk fat yield, milk protein yield, and milk lactose yield (P < 0.001), yet decreased milk urea nitrogen concentration (P = 0.035). Interaction effects of treatment × week were observed for rectal temperature (P < 0.05), respiratory rate (P < 0.05), milk yield (P = 0.015), milk urea nitrogen (P = 0.001), milk protein yield (P = 0.008), and milk lactose yield (P = 0.030). In rumen, LY increased the concentrations of acetate, isobutyrate, isovaterate, valerate, total volatile fatty acids (VFAs), and NH3-N (P < 0.05). Miseq sequencing of the 16S rRNA genes showed that LY increased the relative abundance of Prevotella and Prevotellaceae UCG-003 at the genus level with a series of enriched pathways in the metabolism of carbohydrates and protein. In fecal samples, LY did not affect the profile of VFAs (P > 0.05). Clostridium sensu stricto 1 (P = 0.013) and Actinobacillus (P = 0.011) increased in the relative abundance by LY, whereas Bacteroides (P = 0.016) and Oscillospirales UCG-010 (P = 0.005) decreased with a series of enriched pathways in carbohydrate metabolism, secondary bile acid biosynthesis. In summary, LY supplementation altered the bacterial community's composition and function in rumen and hindgut, and simultaneously alleviated the detrimental effects of heat stress on dairy cows. These findings provide extended insight into the effects of LY in the rumen and hindgut of dairy cows exposed to heat stress.

Dairy cows are exposed to severe heat stress under hot and humid climates in summer in south China, resulting in a decline in feed intake and milk yield. Therefore, we investigated the effect of live yeast (LY, Saccharomyces cerevisiae) supplementation on the milk performance, bacterial community, and functions in the rumen and hindgut of dairy cows under heat stress. Thirty-three dairy cows were randomly assigned to control (CON, without yeast addition), treatment 1 (LY-10, with 10 g yeast/d/head) and treatment 2 (LY-20, with 20 g yeast/d/head). Supplementing LY decreased the rectal temperature and respiratory rate of the dairy cows and increased feed intake and milk performance. Live yeast enhanced fermentation in the rumen but did not affect it in the hindgut. Live yeast altered the microbiota in the rumen and hindgut, with an enrichment of bacteria in the pathways of the metabolism of carbohydrates, protein, and other substances. In all, LY supplementation had beneficial effects on dairy cows under heat stress by affecting the microbiota and fermentation in the rumen and hindgut.

Gene Therapy in Combination with Nitrogen Scavenger Pretreatment Corrects Biochemical and Behavioral Abnormalities of Infant Citrullinemia Type 1 Mice.

Citrullinemia type I (CTLN1) is a rare autosomal recessive disorder caused by mutations in the gene encoding argininosuccinate synthetase 1 (ASS1) that catalyzes the third step of the urea cycle. CTLN1 patients suffer from impaired elimination of nitrogen, which leads to neurotoxic levels of circulating ammonia and urea cycle byproducts that may cause severe metabolic encephalopathy, death or irreversible brain damage. Standard of care (SOC) of CTLN1 consists of daily nitrogen-scavenger administration, but patients remain at risk of life-threatening decompensations. We evaluated the therapeutic efficacy of a recombinant adeno-associated viral vector carrying the ASS1 gene under the control of a liver-specific promoter (VTX-804). When administered to three-week-old CTLN1 mice, all the animals receiving VTX-804 in combination with SOC gained body weight normally, presented with a normalization of ammonia and reduction of citrulline levels in circulation, and 100% survived for 7 months. Similar to what has been observed in CTLN1 patients, CTLN1 mice showed several behavioral abnormalities such as anxiety, reduced welfare and impairment of innate behavior. Importantly, all clinical alterations were notably improved after treatment with VTX-804. This study demonstrates the potential of VTX-804 gene therapy for future clinical translation to CTLN1 patients.

Substrate Recognition Properties from an Intermediate Structural State of the UreA Transporter.

Through a combination of comparative modeling, site-directed and classical random mutagenesis approaches, we previously identified critical residues for binding, recognition, and translocation of urea, and its inhibition by 2-thiourea and acetamide in the Aspergillus nidulans urea transporter, UreA. To deepen the structural characterization of UreA, we employed the artificial intelligence (AI) based AlphaFold2 (AF2) program. In this analysis, the resulting AF2 models lacked inward- and outward-facing cavities, suggesting a structural intermediate state of UreA. Moreover, the orientation of the W82, W84, N279, and T282 side chains showed a large variability, which in the case of W82 and W84, may operate as a gating mechanism in the ligand pathway. To test this hypothesis non-conservative and conservative substitutions of these amino acids were introduced, and binding and transport assessed for urea and its toxic analogue 2-thiourea, as well as binding of the structural analogue acetamide. As a result, residues W82, W84, N279, and T282 were implicated in substrate identification, selection, and translocation. Using molecular docking with Autodock Vina with flexible side chains, we corroborated the AF2 theoretical intermediate model, showing a remarkable correlation between docking scores and experimental affinities determined in wild-type and UreA mutants. The combination of AI-based modeling with classical docking, validated by comprehensive mutational analysis at the binding region, would suggest an unforeseen option to determine structural level details on a challenging family of proteins.

Effects of substituting agro-industrial by-products for soybean meal on beef cattle feed utilization and rumen fermentation.

The purpose of the present investigation was to detect the effect of replacement of soybean meal (SBM) with citric waste fermented yeast waste (CWYW) as an alternative protein source of portentous substances in a concentrate mixture diet of beef cattle on intake, digestibility, ruminal fermentation, plasma urea-nitrogen, energy partitioning, and nitrogen balance. Four Thai-native beef bulls (170 ± 10.0 kg of initial body weight) were randomly allocated to a 4 × 4 Latin square design. The dietary treatments were four levels of CWYW replacing SBM in a concentrated diet at ratios of 0, 33, 67, and 100%. SBM was added to the concentrate diet at a dose of 150 g/kg DM. All cattle were offered ad libitum rice straw and the concentrate diet at 5 g/kg of body weight. The study was composed of four periods, each lasting for 21 days. The findings demonstrated that there was no difference in total dry matter intake, nutritional intake, or digestibility between treatments (p > 0.05). When CWYW replaced SBM at 100% after 4 h of feeding, ruminal pH, ammonia nitrogen, plasma urea nitrogen, and bacterial population were highest (p < 0.05). Volatile fatty acids and energy partitioning were not different (p > 0.05) among dietary treatments. Urinary nitrogen excretion was greatest (p < 0.05) for cattle fed CWYW to replace SBM at 100% of the concentrate. However, nitrogen absorption and retention for Thai-native cattle were similar (p > 0.05) among treatments. In conclusion, CWYW may be utilized as a substitute for SBM as a source of protein in Thai-native beef cattle without having an adverse impact on feed utilization, rumen fermentation characteristics, or blood metabolites.

Influencing factors on ureolytic microbiologically induced calcium carbonate precipitation for biocementation.

Microbiologically induced calcium carbonate precipitation (MICP) is a technique that has received a lot of attention in the field of geotechnology in the last decade. It has the potential to provide a sustainable and ecological alternative to conventional consolidation of minerals, for example by the use of cement. From a variety of microbiological metabolic pathways that can induce calcium carbonate (CaCO3) precipitation, ureolysis has been established as the most commonly used method. To better understand the mechanisms of MICP and to develop new processes and optimize existing ones based on this understanding, ureolytic MICP is the subject of intensive research. The interplay of biological and civil engineering aspects shows how interdisciplinary research needs to be to advance the potential of this technology. This paper describes and critically discusses, based on current literature, the key influencing factors involved in the cementation of sand by ureolytic MICP. Due to the complexity of MICP, these factors often influence each other, making it essential for researchers from all disciplines to be aware of these factors and its interactions. Furthermore, this paper discusses the opportunities and challenges for future research in this area to provide impetus for studies that can further advance the understanding of MICP.

Multiple regulatory mechanisms for pH homeostasis in the gastric pathogen, Helicobacter pylori.

Acid-resistance in gastric pathogen Helicobacter pylori requires the coordination of four essential processes to regulate urease activity. Firstly, urease expression above a base level needs to be finely tuned at different ambient pH. Secondly, as nickel is needed to activate urease, nickel homeostasis needs to be maintained by proteins that import and export nickel ions, and sequester, store and release nickel when needed. Thirdly, urease accessary proteins that activate urease activity by nickel insertion need to be expressed. Finally, a reliable source of urea needs to be maintained by both intrinsic and extrinsic sources of urea. Two-component systems (arsRS and flgRS), as well as a nickel response regulator (NikR), sense the change in pH and act on a variety of genes to accomplish the function of acid resistance without causing cellular overalkalization and nickel toxicity. Nickel storage proteins also feature built-in switches to store nickel at neutral pH and release nickel at low pH. This review summarizes the current status of H. pylori research and highlights a number of hypotheses that need to be tested.

Zinc- and magnesium-doped hydroxyapatite-urea nanohybrids enhance wheat growth and nitrogen uptake.

The ongoing and unrestrained application of nitrogen fertilizer to agricultural lands has been directly linked to climate change and reductions in biodiversity. The agricultural sector needs a technological upgrade to adopt sustainable methods for maintaining high yield. We report synthesis of zinc and magnesium doped and undoped hydroxyapatite nanoparticles, and their urea nanohybrids, to sustainably deliver nitrogen to wheat. The urea nanohybrids loaded with up to 42% nitrogen were used as a new source of nitrogen and compared with a conventional urea-based fertilizer for efficient and sufficient nitrogen delivery to pot-grown wheat. Doping with zinc and magnesium manipulated the hydroxyapatite crystallinity for smaller size and higher nitrogen loading capacity. Interestingly, 50% and 25% doses of urea nanohybrids significantly boosted the wheat growth and yield compared with 100% doses of urea fertilizer. In addition, the nutritional elements uptake and grain protein and phospholipid levels were significantly enhanced in wheat treated with nanohybrids. These results demonstrate the potential of the multi-nutrient complexes, the zinc and magnesium doped and undoped hydroxyapatite-urea nanoparticles, as nitrogen delivery agents that reduce nitrogen inputs by at least 50% while maintaining wheat plant growth and nitrogen uptake to the same level as full-dose urea treatments.

Relationship between different sources of non-protein nitrogen and supplementation times on performance and metabolism of grazing Nellore cattle during the dry season.

Two experiments were conducted to evaluate the effect of supplementation with two sources of non-protein nitrogen at different feeding times on the performance, ingestive behavior, and rumen metabolism of growing Nellore bulls during the dry season. Exp. 1: One hundred and twenty Nellore bulls, weighing 206 ± 39 kg of initial body weight (BW) and 12 months of age, were divided into 20 paddocks, and they were used in randomized block design in a 2 × 2 factorial arrangement to evaluate performance and ingestive behavior. Exp. 2: 12 rumen cannulated animals with 509 ± 59 BW, divided into 4 paddocks, were used in a triple Latin square 4 × 4 in a 2 × 2 factorial arrangement to evaluate metabolism. The factors were 2 non-protein nitrogen sources (urea or slow-release urea) and 2 feeding times (07:00 or 13:00 at 4 g/kg BW of supplement). There was no influence of non-protein sources, supplementation time, or their interaction on the grazing time or the trough time during daytime, nighttime, or total (P ≥ 0.16). There were no interactions or factor effects on ADG (P ≥ 0.45) or final body weight (P ≥ 0.39). There was an interaction between supplementation time and collection time (P < 0.01) on ruminal pH. Animals supplemented in the morning had greater total SCFA at 18 h after supplementation (P = 0.03). The supplementation time and the non-protein nitrogen sources did not alter the ingestive behavior or animal performance of young Nellore cattle.

Two closely related ureotelic fish species of the genus Alcolapia express different levels of ammonium transporters in gills.

Most fish excrete their nitrogenous waste across the gills as ammonia through the activity of the Rhesus glycoprotein ammonium transporters. In contrast, fish of the subgenus Alcolapia (Oreochromis) are the only vertebrates that survive the extreme conditions of the soda lakes of Natron and Magadi in East Africa and have evolved adaptations to the highly alkaline waters including the ability to excrete their nitrogenous waste as urea. Nevertheless, Alcolapia retain the Rhesus glycoprotein genes in their genomes and using two heterologous expression systems, we demonstrate that Alcolapia Rhbg is capable of moving ammonia. Comparing ammonia and urea excretion from two closely related Alcolapia species from the same aquarium, we found that while Alcolapia grahami remains fully ureotelic after many generations in lab conditions, Alcolapia alcalica excretes some of its nitrogenous waste as ammonia. Using in situ hybridisation, we demonstrate robust, localised gene expression of Rhbg, rhcg1 and rhcg2 in the gill tissue in both A. alcalica embryos and adults, similar to that in other ammoniotelic fish. In contrast, the expression of these genes in A. grahami gills is much lower than in A. alcalica, suggesting the rapid evolution of a molecular mechanism underlying the complete ureotelism of A. grahami.

Hepatic urea, creatinine and uric acid metabolism in dairy cows with divergent milk urea concentrations.

Milk urea concentration is an indicator for dietary nitrogen (N)-supply and urinary N-excretion. Dairy cows with high (HMU) compared to low milk urea (LMU) concentration have greater plasma urea, creatinine and uric acid concentrations, but if the liver metabolism accounts for these differences is unknown. Eighteen HMU and 18 LMU cows were fed a diet with a low (LP) or normal (NP) crude protein concentration. A N balance study was performed and a 13C-urea bolus was administered to measure urea pool size. Liver samples were analyzed by 2D-gel-based proteomics and RT-qPCR. Although HMU cows had a greater urea pool, plasma urea, uric acid, and hippuric acid concentrations, these differences were not associated with altered expressions of genes related to urea cycling or N-metabolism. Instead, HMU cows had higher oxidative stress levels. Conclusively, other factors than hepatic urea metabolism account for milk urea concentrations. Despite higher plasma urea concentrations and argininosuccinate synthase 1 protein expression on the LP diet, urea cycle mRNA expressions were not affected, indicating that its activity is not controlled at transcriptional level. Feeding the LP diet resulted in increased expressions of enzymes catabolizing fatty acids, but the reason remains to be investigated in future studies.

Domino hepatocyte transplantation using explanted human livers with metabolic defects attenuates D-GalN/LPS-induced acute liver failure.

BACKGROUND: Explanted livers from patients with inherited metabolic liver diseases possess the potential to be a cell source of good-quality hepatocytes for hepatocyte transplantation (HT). This study evaluated the therapeutic effects of domino HT using hepatocytes isolated from explanted human livers for acute liver failure (ALF). METHODS: Isolated hepatocytes were evaluated for viability and function and then transplanted into D-galactosamine/lipopolysaccharide-induced ALF mice via splenic injection. The survival rate was analyzed by the Kaplan-Meier method and log-rank test. Liver function was evaluated by serum biochemical parameters, and inflammatory cytokine levels were measured by ELISA. The pathological changes in the liver tissues were assessed by hematoxylin-eosin staining. Hepatocyte apoptosis was investigated by TUNEL, and hepatocyte apoptosis-related proteins were detected by western blot. The localization of human hepatocytes in the injured mouse livers was detected by immunohistochemical analyses. RESULTS: Hepatocytes were successfully isolated from explanted livers of 10 pediatric patients with various liver-based metabolic disorders, with an average viability of 85.3% ± 13.0% and average yield of 9.2 × 106 ± 3.4 × 106 cells/g. Isolated hepatocytes had an excellent ability to secret albumin, produce urea, uptake indocyanine green, storage glycogen, and express alpha 1 antitrypsin, albumin, cytokeratin 18, and CYP3A4. Domino HT significantly reduced mortality, decreased serum levels of alanine aminotransferase and aspartate aminotransferase, and improved the pathological damage. Moreover, transplanted hepatocytes inhibited interleukin-6 and tumor necrosis factor-α levels. Domino HT also ameliorates hepatocyte apoptosis, as evidenced by decreased TUNEL positive cells. Positive staining for human albumin suggested the localization of human hepatocytes in ALF mice livers. CONCLUSION: Explanted livers from patients with inheritable metabolic disorders can serve as a viable cell source for cell-based therapies. Domino HT using hepatocytes with certain metabolic defects has the potential to be a novel therapeutic strategy for ALF.

Divergent Pathways of Ammonia and Urea Production and Excretion during the Life Cycle of the Sea Lamprey.

Little is known about nitrogenous waste (N waste) handling and excretion (JN waste) during the complex life cycle of the sea lamprey (Petromyzon marinus), an extant jawless fish that undergoes a complete metamorphosis from a filter-feeding larva (ammocoete) into a parasitic juvenile that feeds on the blood of larger, jawed fishes. Here, we investigate the ammonia- and urea-handling profiles of sea lampreys before, during, and after metamorphosis. The rates of ammonia excretion (Jamm) and urea excretion (Jurea) significantly decreased after the onset of metamorphosis, with the lowest rates observed during midmetamorphosis. Near the completion of metamorphosis, rates of JN waste (JN waste=Jamm+Jurea) significantly increased as sea lampreys entered the juvenile period. Feeding juvenile lampreys had greater than 10- to 15-fold higher Jamm and fivefold higher Jurea compared to nonfed juveniles, which corresponded to higher postprandial (postfeeding) concentrations of plasma ammonia and urea. The routes of Jamm and Jurea completely diverged following metamorphosis. In larvae, Jamm was equally split between branchial (gills) and extrabranchial (skin plus renal) pathways, but following metamorphosis, >80% of ammonia was excreted via the gills in nonfeeding juvenile lampreys, and >95% of ammonia was excreted via the gills in adult sea lampreys. Urea, on the other hand, was predominantly excreted via extrabranchial routes and, to a lesser extent, the gills in larvae and in nonfeeding juveniles. In adults, however, virtually all urea was excreted via urine. Reverse transcription polymerase chain reaction and in silico analyses also indicated that a urea transporter encoded by a slc4a2-like gene is present in lampreys. The branchial expression of this transporter is modulated throughout sea lamprey life history, as it is higher in the larvae and steadily decreases until the adult stage. We conclude that the divergent pathways of Jamm and Jurea during the sea lamprey life cycle reflect changes in their habitat, lifestyle, and diet. Further, the near-complete reliance on renal routes for Jurea in adult sea lampreys is unique among fishes and may reflect the ancestral condition of how this N waste product was handled and excreted by the earliest vertebrates.

Helicobacter pylori shows tropism to gastric differentiated pit cells dependent on urea chemotaxis.

The human gastric epithelium forms highly organized gland structures with different subtypes of cells. The carcinogenic bacterium Helicobacter pylori can attach to gastric cells and subsequently translocate its virulence factor CagA, but the possible host cell tropism of H. pylori is currently unknown. Here, we report that H. pylori preferentially attaches to differentiated cells in the pit region of gastric units. Single-cell RNA-seq shows that organoid-derived monolayers recapitulate the pit region, while organoids capture the gland region of the gastric units. Using these models, we show that H. pylori preferentially attaches to highly differentiated pit cells, marked by high levels of GKN1, GKN2 and PSCA. Directed differentiation of host cells enable enrichment of the target cell population and confirm H. pylori preferential attachment and CagA translocation into these cells. Attachment is independent of MUC5AC or PSCA expression, and instead relies on bacterial TlpB-dependent chemotaxis towards host cell-released urea, which scales with host cell size.

Discerning Comparison of 1 and 0.5% Ethylene Glycol in Sprague-Dawley Rats with Modeled Urolithiasis.

A common method of modeling urolithiasis is the use of 1 and 0.75% ethylene glycol, or a combination of ethylene glycol with other lithogens, but too rapid progression of the disease and multiple organ toxicity have been reported. We developed a urolithiasis model in Sprague-Dawley rats, in which the animals received a relatively low concentration of ethylene glycol (0.5%), but for a long-term period (6 weeks) followed by animal observation during the 6-week recovery period. In urine samples, signs of the urolithiasis development were observed starting from the sixth week: the presence of ketones, decrease in diuresis and urine pH; in the blood, urea, protein, and hematocrit were elevated. However, no leukocytes were detected in the urine; in the blood, no shifts in differential leukocyte count and no elevation in ALT, creatinine, cholesterol, and triglycerides were observed, which indicates the absence of multiple organ failure while using 1% ethylene glycol. In addition, the animals receiving 0.5% ethylene glycol were followed up to 12 weeks in contrast to animals receiving 1% ethylene glycol (the experiment in this case was stopped during the third week for ethical reasons).