ABCG5/G8 Crystallization in a Lipidic Bicelle Environment for X-Ray Crystallography.

ATP-binding cassette (ABC) transporters constitute lipid-embedded membrane proteins. Extracting these membrane proteins from the lipid bilayer to an aqueous environment is typically achieved by employing detergents. These detergents disintegrate the lipid bilayer and solubilize the proteins. The intrinsic habitat of membrane proteins within the lipid bilayer poses a challenge in maintaining their stability and uniformity in solution for structural characterization. Bicelles, which comprise a blend of long and short-chain phospholipids and detergents, replicate the natural lipid structure. The utilization of lipid bicelles and detergents serves as a suitable model system for obtaining high-quality diffraction crystals, specifically to determine the high-resolution structure of membrane proteins. Through these synthetic microenvironments, membrane proteins preserve their native conformation and functionality, facilitating the formation of three-dimensional crystals. In this approach, the detergent-solubilized heterodimeric ABCG5/G8 was reintegrated into DMPC/CHAPSO bicelles, supplemented with cholesterol. This setup was employed in the vapor diffusion experimental procedure for protein crystallization.

W546 stacking disruption traps the human porphyrin transporter ABCB6 in an outward-facing transient state.

Human ATP-binding cassette transporter subfamily B6 (ABCB6) is a mitochondrial ATP-driven pump that translocates porphyrins from the cytoplasm into mitochondria for heme biosynthesis. Within the transport pathway, a conserved aromatic residue W546 located in each monomer plays a pivotal role in stabilizing the occluded conformation via π-stacking interactions. Herein, we employed cryo-electron microscopy to investigate the structural consequences of a single W546A mutation in ABCB6, both in detergent micelles and nanodiscs. The results demonstrate that the W546A mutation alters the conformational dynamics of detergent-purified ABCB6, leading to entrapment of the transporter in an outward-facing transient state. However, in the nanodisc system, we observed a direct interaction between the transporter and a phospholipid molecule that compensates for the absence of the W546 residue, thereby facilitating the normal conformational transition of the transporter toward the occluded state following ATP hydrolysis. The findings also reveal that adoption of the outward-facing conformation causes charge repulsion between ABCB6 and the bound substrate, and rearrangement of key interacting residues at the substrate-binding site. Consequently, the affinity for the substrate is significantly reduced, facilitating its release from the transporter.

Polyamine detergents tailored for native mass spectrometry studies of membrane proteins.

Native mass spectrometry (MS) is a powerful technique for interrogating membrane protein complexes and their interactions with other molecules. A key aspect of the technique is the ability to preserve native-like structures and noncovalent interactions, which can be challenging depending on the choice of detergent. Different strategies have been employed to reduce charge on protein complexes to minimize activation and preserve non-covalent interactions. Here, we report the synthesis of a class of polyamine detergents tailored for native MS studies of membrane proteins. These detergents, a series of spermine covalently attached to various alkyl tails, are exceptional charge-reducing molecules, exhibiting a ten-fold enhanced potency over spermine. Addition of polyamine detergents to proteins solubilized in maltoside detergents results in improved, charge-reduced native mass spectra and reduced dissociation of subunits. Polyamine detergents open new opportunities to investigate membrane proteins in different detergent environments that have thwarted previous native MS studies.

Biomass yield, quality, nutrient composition, and feeding value of oat (Avena sativa) silage subjected to different wilting durations and/or inoculant application.

In this study, we evaluated the biomass yield, physico-chemical characteristics, nutrient composition, and feeding value of oat (Avena sativa) grown without irrigation ensiled with or without supplemental inoculant following different wilting durations. Oat forage at early dough stage (79 days after sowing) were harvested to assess the biomass yield, nutrient contents, and mineral composition. Oats were ensiled with or without the addition of inoculant and different wilting durations (0, 24, and 48 h) in 3 × 2 factorial arrangement. After the ensiling (120 days), the oat silages were opened, quality was measured in terms of pH, ammonia nitrogen (NH3-N), smell, structure, color, and Flieg point. Nutritional composition and feeding values were analyzed in oat silages. Oat grown without irrigation yielded 32 ton/ha fresh matter. Mean dry matter (DM), organic matter, crude protein (CP), crude fiber, crude ash, ether extract, nitrogen free extract, acid detergent fiber (ADF), neutral detergent fiber, acid detergent lignin, non-structural carbohydrates, hemicellulose, and in vitro dry matter digestibility of oat forage were 32.77%, 90.41%, 11.31%, 28.69%, 9.59%, 3.99%, 46.43%, 36.32%, 63.98%, 7.22%, 11.14%, 27.67%, and 74.81%, respectively. Addition of inoculant had no effect on the quality, nutritional composition, and feeding values of oat silages. Increasing wilting durations linearly increased the pH (P = 0.005) and decreased the smell score (P = 0.028) of ensiled oat. A linear increase was seen in the DM content of ensiled oat after increasing wilting durations (P = 0.001). Oat ensiled without wilting had greater CP content (P = 0.010 and linear P = 0.011) and lower ADF content than those ensiled after 24 or 48 h of wilting (P = 0.013 and linear P = 0.007). Silages subjected to 24 or 48 h of wilting had lower hemicellulose content (P = 0.019 and linear P = 0.012) and digestible DM (P = 0.013 and linear P = 0.007) than those without wilting. In conclusion, inoculant may not affect the quality, composition and feeding values of ensiled oat grown without irrigation whereas, wilting at different durations may negatively affect the pH, smell, CP, ADF, and feeding values of ensiled oats.

Metabolic responses and performance of Holstein × Gyr heifers grazing Brachiaria decumbens supplemented with varied crude protein levels.

We aimed to evaluate the effect of supplemental CP on the nutritional characteristics and performance of Holstein × Gyr crossbreed heifers grazing intensively-managed Brachiaria decumbens throughout the year. Thirty-eight heifers with average initial body weight of 172.5 ± 11.15 kg (mean ± SE) and 8.2 ± 0.54 mo of age were randomly assigned to four treatments: three protein supplements (SUP) composed of soybean meal and ground corn fed at 5g/kg of BW, plus a control group (CON). The supplements had 12, 24 and 36% of CP for treatments S12 (n = 9), S24 (n = 10), and S36 (n = 9), respectively. The experiment latest one year, subdivided into four seasons: rainy, dry, rainy-dry transition (RDT), and dry-rainy transition (DRT). Feces and pasture samples were collected for 4 days in each season, using chromium oxide, titanium dioxide, and indigestible neutral detergent fiber (NDF) to estimate fecal excretion, supplement, and pasture intake, respectively. The data were analyzed using PROC GLIMMIXED of the SAS with repeated measures. No effects of supplementation were detected on pasture and NDF intake. However, SUP animals had a greater intake of DM, metabolizable energy, and metabolizable protein. A positive linear response on metabolizable protein intake was observed among SUP animals. We observed an interaction between treatment and season for all digestibility variables, with a positive linear response in CP digestibility among SUP animals during all seasons. For neutral detergent fiber (NDF) digestibility, we observed a positive linear response in RDT and rainy seasons and a quadratic response during the dry season. Furthermore, SUP animals had greater average daily gain (ADG) than non-supplemented animals, and among SUP animals, there was a quadratic response to ADG, with the greatest gain observed in S24. We observed greater nitrogen retention coefficient in SUP animals than in non-supplemented animals and a positive linear effect among SUP animals. Supplemental CP did not affect microbial protein production and efficiency. We observed an interaction between treatment and season for blood glucose, with SUP animals having greater glucose concentration in all seasons than non-supplemented animals. Additionally, we observed a quadratic response among SUP animals only during RDT and dry season, with the greatest glucose concentration in S24. SUP animals had greater blood concentrations of urea and IGF-1. In conclusion, SUP animals had greater intake, digestibility, and performance than non-supplemented animals, with the 24% CP supplement demonstrating the best metabolic responses and performance.

Phospholipid headgroup composition modulates the molecular interactions and antimicrobial effects of sulfobetaine zwitterionic detergents against the "ESKAPE" pathogen Pseudomonas aeruginosa.

We determine the efficacy for three known structurally related, membrane active detergents against multidrug resistant and wild type strains of Pseudomonas aeruginosa. Accessible solution state NMR experiments are used to quantify phospholipid headgroup composition of the microbial membranes and to gain molecular level insight into antimicrobial mode of action.

Infrared Photoactivation Enables Improved Native Top-Down Mass Spectrometry of Transmembrane Proteins.

Membrane proteins are often challenging targets for native top-down mass spectrometry experimentation. The requisite use of membrane mimetics to solubilize such proteins necessitates the application of supplementary activation methods to liberate protein ions prior to sequencing, which typically limits the sequence coverage achieved. Recently, infrared photoactivation has emerged as an alternative to collisional activation for the liberation of membrane proteins from surfactant micelles. However, much remains unknown regarding the mechanism by which IR activation liberates membrane protein ions from such micelles, the extent to which such methods can improve membrane protein sequence coverage, and the degree to which such approaches can be extended to support native proteomics. Here, we describe experiments designed to evaluate and probe infrared photoactivation for membrane protein sequencing, proteoform identification, and native proteomics applications. Our data reveal that infrared photoactivation can dissociate micelles composed of a variety of detergent classes, without the need for a strong IR chromophore by leveraging the relatively weak association energies of such detergent clusters in the gas phase. Additionally, our data illustrate how IR photoactivation can be extended to include membrane mimetics beyond micelles and liberate proteins from nanodiscs, liposomes, and bicelles. Finally, our data quantify the improvements in membrane protein sequence coverage produced through the use of IR photoactivation, which typically leads to membrane protein sequence coverage values ranging from 40 to 60%.

Susceptibility of Cu(I) transport ATPases to sodium dodecyl sulfate. Relevance of the composition of the micellar phase.

Sodium dodecyl sulfate (SDS) is a well-known protein denaturing agent. A less known property of this detergent is that it can activate or inactivate some enzymes at sub-denaturing concentrations. In this work we explore the effect of SDS on the ATPase activity of a hyper-thermophilic and a mesophilic Cu(I) ATPases reconstituted in mixed micelles of phospholipids and a non-denaturing detergent. An iterative procedure was used to evaluate the partition of SDS between the aqueous and the micellar phases, allowing to determine the composition of micelles prepared from phospholipid/detergent mixtures. The incubation of enzymes with SDS in the presence of different amounts of phospholipids reveals that higher SDS concentrations are required to obtain the same degree of inactivation when the initial concentration of phospholipids is increased. Remarkably, we found that, if represented as a function of the mole fraction of SDS in the micelle, the degree of inactivation obtained at different amounts of amphiphiles converges to a single inactivation curve. To interpret this result, we propose a simple model involving active and inactive enzyme molecules in equilibrium. This model allowed us to estimate the Gibbs free energy change for the inactivation process and its derivative with respect to the mole fraction of SDS in the micellar phase, the latter being a measure of the susceptibility of the enzyme to SDS. Our results showed that the inactivation free energy changes are similar for both proteins. Conversely, susceptibility to SDS is significantly lower for the hyperthermophilic ATPase, suggesting an inverse relation between thermophilicity and susceptibility to SDS.

Modulation of feed digestibility, nitrogen metabolism, energy utilisation and serum biochemical indices by dietary Ligularia virgaurea supplementation in Tibetan sheep.

Ligularia virgaurea is the most widely functional native herbage in the alpine meadow pastures of the Qinghai-Tibet Plateau (QTP) and has multiple pharmacological and biological activities. The effect of L. virgaurea as a dietary component on the digestion and metabolism of sheep was evaluated by conducting feeding trials in metabolic cages. Thirty-two Tibetan yearling rams (29 ± 1.56 kg BW) were randomly allotted to four groups included in a completely randomised design with eight animals per treatment. Sheep were fed a basal diet (freshly native pasture) without the addition of L. virgaurea (control) or with the addition of L. virgaurea (100, 200, or 300 mg/kg BW per day) for 45 days. Addition of L. virgaurea to the diet of Tibetan sheep was found to influence the average daily gain (quadratic [Q], P < 0.001), feed conversion ratio (Q, P = 0.002), CH4 emissions (linear [L], P = 0.029), DM (Q, P = 0.012), neutral detergent fibre (Q, P = 0.017), acid detergent fibre (ADF) (Q, P = 0.027), and ether extract (EE) intake (Q, P = 0.026). Apparently, different levels of L. virgaurea affected the digestibility coefficients of DM, ADF, and EE (L, P > 0.05; Q, P < 0.05). The nitrogen (N) intake (Q, P = 0.001), retained nitrogen (Q, P < 0.001), and N utilisation efficiency (L, P > 0.05; Q, P ≤ 0.001) were also affected by the dietary inclusion of L. virgaurea. Effects of L. virgaurea feeding were also witnessed on methane energy (CH4-E) (L, P = 0.029), gross energy (GE) (Q, P = 0.013), digestible energy (DE) (Q, P = 0.015), and metabolisable energy (ME) intake (Q, P = 0.015). Energy utilisation efficiency expressed as a proportion of GE intake (DE/GE intake, ME/GE intake, ME/DE intake, FE/GE intake, and CH4-E/GE intake) manifested quadratic changes (P < 0.05) with the increase in the L. virgaurea supplementation level. The addition of L. virgaurea increased the activity of superoxide dismutase (Q, P = 0.026) and glutathione peroxidase activity (Q, P = 0.039) in the serum. Overall, the greatest improvement of feed digestibility, N retention, energy utilisation, and antioxidant capacity of Tibetan sheep was yielded by the inclusion of 200 mg/kg BW per day of L. virgaurea. Therefore, the addition of an appropriate amount of L. virgaurea to the diet of Tibetan sheep is safe and natural, and may enhance the sustainability of small ruminant production systems in QTP areas.

Phospholipid cofactor solubilization inhibits formation of native prions.

Cofactor molecules are required to generate infectious mammalian prions in vitro. Mouse and hamster prions appear to have different cofactor preferences: Whereas both mouse and hamster prions can use phosphatidylethanolamine (PE) as a prion cofactor, only hamster prions can also use single-stranded RNA as an alternative cofactor. Here, we investigated the effect of detergent solubilization on rodent prion formation in vitro. We discovered that detergents that can solubilize PE (n-octylglucoside, n-octylgalactoside, and CHAPS) inhibit mouse prion formation in serial protein misfolding cyclic amplification (sPMCA) reactions using bank vole brain homogenate substrate, whereas detergents that are unable to solubilize PE (Triton X-100 and IPEGAL) have no effect. For all three PE-solubilizing detergents, inhibition of RML mouse prion formation was only observed above the critical micellar concentration (CMC). Two other mouse prion strains, Me7 and 301C, were also inhibited by the three PE-solubilizing detergents but not by Triton X-100 or IPEGAL. In contrast, none of the detergents inhibited hamster prion formation in parallel sPMCA reactions using the same bank vole brain homogenate substrate. In reconstituted sPMCA reactions using purified substrates, n-octylglucoside inhibited hamster prion formation when immunopurified bank vole PrPC substrate was supplemented with brain phospholipid but not with RNA. Interestingly, phospholipid cofactor solubilization had no effect in sPMCA reactions using bacterially expressed recombinant PrP substrate, indicating that the inhibitory effect of solubilization requires PrPC post-translational modifications. Overall, these in vitro results show that the ability of PE to facilitate the formation of native but not recombinant prions requires phospholipid bilayer integrity, suggesting that membrane structure may play an important role in prion formation in vivo.

Solvents and detergents compatible with enzyme kinetic studies of cholinesterases.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are enzymes that serve a wide range of physiological functions including the hydrolysis of the neurotransmitter acetylcholine and several other xenobiotics. The development of inhibitors for these enzymes has been the focus for the treatment of several conditions, such as Alzheimer's disease. Novel chemical entities are evaluated as potential inhibitors of AChE and BChE using enzyme kinetics. A common issue encountered in these studies is low aqueous solubility of the possible inhibitor. Additives such as cosolvents or detergents can be included in these studies improve the aqueous solubility. Typical cosolvents include acetonitrile or dimethyl sulfoxide while typical detergents include Polysorbate 20 (Tween 20) or 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS). When solubility is not improved, these molecules are often not evaluated further. To address this issue eleven cosolvents and six detergents that could facilitate aqueous solubility were evaluated to understand how they would affect cholinesterase enzymes using Ellman's assay. These studies show that propylene glycol, acetonitrile, methanol, Tween 20, Polysorbate 80 (Tween 80), polyoxyethylene 23 lauryl ether (Brij 35) and polyoxyethylene 10 oleoyl ether (Brij 96v) have the least inhibitory effects towards cholinesterase activity. It is concluded that these cosolvents and detergents should be considered as solubilizing agents for evaluation of potential cholinesterase inhibitors with low aqueous solubility.

Bamboo forage in Peruvian Amazon: a potential feed for cattle.

During the dry and rainy seasons of the Northeastern Zone of Peru, a chemical characterization of five species of bamboo prevalent in the area (Guadua lynnclarkiae, G. takahashiae, Bambusa vulgaris, G. weberbaueri, and Dendrocalamus asper) was conducted. Then, the effect of supplementing bamboo leaves (0, 20, and 40% inclusion of D. asper) on the intake and live weight gain of 18 Gyr × Holstein heifers was evaluated for 28 days. Among the species evaluated, D. asper has the greatest crude protein (CP) concentration (158-166 g/kg Dry matter- DM), post-ruminal CP supply (127 g/kg DM), and in vitro organic matter digestibility (444-456 g/kg DM) but similar concentrations of crude ash (124 g/kg DM), calcium (2.4-2.8 mg/g), phosphorus (0.7-2.1 mg/g), protein fractions A, B1, B2, B3, C (45, 5, 35, 56, and 17g/kg DM, respectively), rumen-undegraded CP (31% CP), neutral detergent fiber (NDF, 685g/kg DM), and acid detergent fiber (ADF, 357 g/kg DM) than the other species evaluated. Dry matter intake was higher in the control treatment and in the 20% bamboo leaf inclusion treatment than in the 40% bamboo inclusion treatment. Intake of CP and NDF decreased with the increase in bamboo inclusion. Despite the differences in DM, CP, and NDF intake, the live weight gain remained similar across treatments. However, there was a greater feed conversion in the 20% bamboo leaf inclusion treatment. During the dry season, bamboo leaves can be used as an alternative supplement at a maximum inclusion of 20% without affecting the live weight gain.

Morpho-physiological traits and SSR markers-based analysis of relationships and genetic diversity among fodder maize landraces in India.

BACKGROUND: Maize is an excellent fodder crop due to its high biomass, better palatability, succulency, and nutrition. Studies on morpho-physiological and biochemical characterization of fodder maize are limited. The present study aimed to explore the genetic variation in fodder maize landraces for various morpho-physiological traits and estimation of genetic relationship and population structure. METHODS AND RESULTS: The study on 47 fodder maize landraces revealed significant variation for all morpho-physiological traits except leaf-stem ratio. Plant height, stem girth, leaf-width and number of leaves showed positive correlation with green fodder yield. Morpho-physiological traits-based clustering grouped the landraces into three major clusters, whereas neighbour joining cluster and population structure analysis using 40 SSR markers revealed four and five major groups, respectively. Most landraces of Northern Himalaya-Kashmir and Ludhiana fall into a single group, whereas rest groups mainly had landraces from North-Eastern Himalaya. A total of 101 alleles were generated with mean polymorphic information content value of 0.36 and major allele frequency of 0.68. The pair wise genetic dissimilarity between genotypes ranged from 0.21 to 0.67. Mantel test revealed weak but significant correlation between morphological and molecular distance. Biochemical characterisation of superior landraces revealed significant variation for neutral detergent fibre, acid detergent fibre, cellulose and lignin content. CONCLUSION: Interestingly, significant, and positive correlation of SPAD with lignin content can be explored to bypass the costly affair of invitro quality assessment for digestibility parameters. The study identified superior landraces and demonstrated the use of molecular markers in genetic diversity assessment and grouping of genotypes for fodder maize improvement.

Multimetal bioremediation from aqueous solution using dead biomass of Mucor sp. NRCC6 derived from detergent manufacturing effluent.

Among ten metal-tolerant fungal isolates obtained from the microbiomes of detergent industry effluent, Mucor sp. NRCC6 showed the highest tolerance and an adaptive behavior toward the heavy metals Ni2+, Pb2+, Mn2+, and Zn2+. It gave the highest growth rates 0.790 ± 0.59, 0.832 ± 0.32, 0.774 ± 0.40, and 0.741 ± 1.06 mm/h along with the lowest growth inhibition 9.19, 4.37, 11.04, and 14.83% in the presence of Pb2+, Zn2+, Ni2+, and Mn2+, respectively, at a concentration of 5.0 g/L. Then, Mucor sp. NRCC6 was selected as a biotrap for the removal of these heavy metals. The optimized operating conditions were detected to be pH 6.0 for Pb2+, Zn2+, and Mn2+ and pH 5.5 for Ni2+ at 30 °C; agitation speed 150 rpm; contact time 30 min for Mn2+ and Ni2+, 30-60 min for Pb2+, and 90-180 min for Zn2+; NRCC6 biomass dosage 5.0 g/L for Ni2+ and Pb2+ and 10.0 g/L for Mn2+ and Zn2+; and initial concentration 12 mg/L of each ion in the multimetal aqueous solutions. Under these optimized conditions, the adsorption capacity for Pb2+, Ni2+, Mn2+, and Zn2+ reached 98.75, 59.25, 58.33, and 50.83%. The Langmuir isotherm was the best for describing the adsorption of Zn2+ (0.970) and Mn2+ (0.977). The Freundlich isotherm significantly giving a good fit to the adsorption of Pb2+ (0.998) while the adsorption of Ni2+ onto NRCC6 biomass can follow DKR (0.998). Furthermore, the current study revealed that Mucor sp. NRCC6 fungus is a new efficient and eco-friendly method that revealed a maximum removal of 100% for Pb2+ and Zn2+ as well as 97.39, 88.70, 78.95, 74.0, 70.22, 68.57, and 60.0% for Ni2+, Mn2+, Cd2+, Cu2+, Fe3+, As2+, and Cr6+ from the industrial wastewater, respectively.

On-column virus inactivation by solvent/detergent treatment for a recombinant biological product.

Each process step in the manufacture of biological products requires expensive resources and reduces total process productivity. Since downstream processing of biologicals is the main cost driver, process intensification is a persistent topic during the entire product life cycle. We present here one approach for the intensification of bioprocesses by applying on-column virus inactivation using solvent/detergent (S/D) treatment during ion-exchange chromatography. The established purification process of a recombinant protein was used as a model to compare key process parameters (i.e., product yield, specific activity, impurity clearance) of the novel approach to the standard process protocol. Additional wash and incubation steps with and without S/D-containing buffers were introduced to ensure sufficient contact time to effectively eliminate enveloped viruses and to significantly decrease the amount of S/D reagents. Comparison of key process parameters demonstrated equivalent process performance. To assess the viral clearance capacity of the novel approach, XMuLV was spiked as model virus to the chromatographic load and all resulting fractions were analyzed by TCID50 and RT-qPCR. Data indicates the inactivation capability of on-column virus inactivation even at 10% of the nominal S/D concentration, although the mechanism of viral clearance needs further investigation.

Structures and membrane interactions of native serotonin transporter in complexes with psychostimulants.

The serotonin transporter (SERT) is a member of the SLC6 neurotransmitter transporter family that mediates serotonin reuptake at presynaptic nerve terminals. SERT is the target of both therapeutic antidepressant drugs and psychostimulant substances such as cocaine and methamphetamines, which are small molecules that perturb normal serotonergic transmission by interfering with serotonin transport. Despite decades of studies, important functional aspects of SERT such as the oligomerization state of native SERT and its interactions with potential proteins remain unresolved. Here, we develop methods to isolate SERT from porcine brain (pSERT) using a mild, nonionic detergent, utilize fluorescence-detection size-exclusion chromatography to investigate its oligomerization state and interactions with other proteins, and employ single-particle cryo-electron microscopy to elucidate the structures of pSERT in complexes with methamphetamine or cocaine, providing structural insights into psychostimulant recognition and accompanying pSERT conformations. Methamphetamine and cocaine both bind to the central site, stabilizing the transporter in an outward open conformation. We also identify densities attributable to multiple cholesterol or cholesteryl hemisuccinate (CHS) molecules, as well as to a detergent molecule bound to the pSERT allosteric site. Under our conditions of isolation, we find that pSERT is best described as a monomeric entity, isolated without interacting proteins, and is ensconced by multiple cholesterol or CHS molecules.

Production effects of extruded soybean meal replacing canola meal in the diet of lactating dairy cows.

This study investigated the effects of extruded soybean meal (ESBM) in comparison with canola meal (CM) fed on an equivalent crude protein (CP) basis on lactational performance and ruminal fermentation of dairy cows. Following a 2-wk covariate period, 48 Holstein cows averaging (±SD): 146 ± 46 d in milk (DIM) and 43 ± 7 kg/d milk yield (MY) were assigned 1 of 2 treatment diets in a randomized complete block design experiment, which included a 2-wk period for dietary treatment adaptation before experimental data were collected. Following the adaptation period, samples and experimental data were collected for a total of 7 wk. Cows were blocked based on parity, DIM, and MY. Treatment diets contained 15.8% CM (containing 41.2% CP) or 13.2% ESBM (with 48.7% CP) of total mixed ration dry matter (DM), with similar inclusion of other feed ingredients. The CM diet was supplemented with canola oil, whereas the ESBM diet was supplemented with soybean hulls to achieve similar ether extract and neutral detergent fiber contents between the diets. Urea and rumen-protected Met and Lys were added to both diets to meet or exceed cow recommendations. Whole-ruminal digesta samples were collected from 10 (5 per treatment) ruminally cannulated cows. Eight cannulated cows were removed during the last week of the experiment to participate in another study. Treatment did not affect DM intake and MY or energy-corrected MY of the cows. Energy-corrected MY, apart from experimental wk 5, was similar between treatments. Apart from experimental wk 3 and 7, milk fat concentration and yield were greater for cows fed ESBM compared with CM. In multiparous cows only, milk true protein yield was greater for cows fed CM compared with ESBM. Ruminal concentration of total volatile fatty acids and the molar proportion of acetate were greater for ESBM, and propionate and valerate were greater in cows fed CM. Acetate to propionate ratio was greater for cows fed ESBM versus CM diet. Compared with the CM diet, the ESBM diet increased plasma concentrations of Ile, Leu, and Phe but not the sum of essential AA. Apparent total-tract digestibility of acid detergent fiber was greater in cows fed ESBM relative to CM. In this experiment, CM and ESBM included on an equal CP basis in the diet of dairy cows, resulted in similar DM intake, MY, and feed efficiency.

Perfusion and Ultrasonication Produce a Decellularized Porcine Whole-Ovary Scaffold with a Preserved Microarchitecture.

The application of decellularized scaffolds for artificial tissue reconstruction has been an approach with great therapeutic potential in regenerative medicine. Recently, biomimetic ovarian tissue reconstruction was proposed to reestablish ovarian endocrine functions. Despite many decellularization methods proposed, there is no established protocol for whole ovaries by detergent perfusion that is able to preserve tissue macro and microstructure with higher efficiency. This generated biomaterial may have the potential to be applied for other purposes beyond reproduction and be translated to other areas in the tissue engineering field. Therefore, this study aimed to establish and standardize a protocol for porcine ovaries' decellularization based on detergent perfusion and ultrasonication to obtain functional whole-ovary scaffolds. For that, porcine ovaries (n = 5) were perfused with detergents (0.5% SDS and 1% Triton X-100) and submitted to an ultrasonication bath to produce acellular scaffolds. The decellularization efficiency was evaluated by DAPI staining and total genomic DNA quantification. ECM morphological evaluation was performed by histological, immunohistochemistry, and ultrastructural analyses. ECM physico-chemical composition was evaluated using FTIR and Raman spectroscopy. A cytocompatibility and cell adhesion assay using murine fibroblasts was performed. Results showed that the proposed method was able to remove cellular components efficiently. There was no significant ECM component loss in relation to native tissue, and the scaffolds were cytocompatible and allowed cell attachment. In conclusion, the proposed decellularization protocol produced whole-ovaries scaffolds with preserved ECM composition and great potential for application in tissue engineering.

Solvent/detergent treated pooled human plasma can decrease the recurrence of allergic transfusion reactions in pediatric, adolescent, and young adult patients.

BACKGROUND: Octaplas is a solvent/detergent (S/D)-treated pooled human plasma indicated for the treatment of thrombotic thrombocytopenic purpura (TTP) as well as multiple coagulation factor deficiency in patients with liver disease or undergoing liver transplantation or cardiac surgery. We aimed at providing pediatric, adolescent, and young adult evidence for the decrease in allergic transfusion reactions (ATRs) with S/D-treated plasma. STUDY DESIGN/METHODS: A single-center retrospective review of patient records was performed from January 2018 through July 2022 for patients who received S/D treated plasma (Octaplas™; Octapharma). RESULTS/FINDINGS: A total of 1415 units of S/D-treated plasma were transfused to nine patients at our institution. Patient ages ranged from 13 months to 25 years old. The reason to initiate transfusion with S/D treated plasma in six patients was mild to severe ATR to plasma-containing products and the need for therapeutic plasma exchange (TPE) or plasma transfusions (PTs). TPE or PT was performed for various clinical indications. Average S/D treated plasma volume per TPE or PT ranged from 200 to 1800 mL per event. During the study period, since initiating transfusions with S/D treated plasma, there have been no allergic or other transfusion reactions reported among these patients. CONCLUSION: We have successfully utilized S/D treated plasma over the last 4.5 years for pediatric, adolescent, and young adult patients who otherwise would have suffered ATR due to necessary TPE or PT. S/D treated plasma is an additional tool that can be utilized by transfusion services, including pediatrics, to safely transfuse their patients.

Infrared Multiphoton Dissociation Enables Top-Down Characterization of Membrane Protein Complexes and G Protein-Coupled Receptors.

Membrane proteins are challenging to analyze by native mass spectrometry (MS) as their hydrophobic nature typically requires stabilization in detergent micelles that are removed prior to analysis via collisional activation. There is however a practical limit to the amount of energy which can be applied, which often precludes subsequent characterization by top-down MS. To overcome this barrier, we have applied a modified Orbitrap Eclipse Tribrid mass spectrometer coupled to an infrared laser within a high-pressure linear ion trap. We show how tuning the intensity and time of incident photons enables liberation of membrane proteins from detergent micelles. Specifically, we relate the ease of micelle removal to the infrared absorption of detergents in both condensed and gas phases. Top-down MS via infrared multiphoton dissociation (IRMPD), results in good sequence coverage enabling unambiguous identification of membrane proteins and their complexes. By contrasting and comparing the fragmentation patterns of the ammonia channel with two class A GPCRs, we identify successive cleavage of adjacent amino acids within transmembrane domains. Using gas-phase molecular dynamics simulations, we show that areas prone to fragmentation maintain aspects of protein structure at increasing temperatures. Altogether, we propose a rationale to explain why and where in the protein fragment ions are generated.