Decreased Protein Degradation Research Articles

Knockout of PA200 improves proteasomal degradation and myelination in a proteotoxic neuropathy.

The cellular response to a decrease in protein degradation by 26S proteasomes in chronic diseases is poorly understood. Pharmacological inhibition of proteasomes increases the expression of proteasome subunits and Proteasome Activator 200 (PA200), an alternative proteasome activator. In the S63del mouse model of the peripheral neuropathy Charcot Marie Tooth 1B (CMT1B), proteasomal protein degradation is decreased and proteasome gene expression is increased. Here, we show an increase in PA200 and PA200-bound proteasomes in the peripheral nerves of S63del mice. To test genetically whether the upregulation of PA200 was compensatory, we generated S63del//PA200-/- mice. Unexpectedly, in the sciatic nerves of these mice, there was greater proteasomal protein degradation than in S63del, less polyubiquitinated proteins and markers of the unfolded protein response, and a greater amount of assembled, active 26S proteasomes. These changes were not seen in PA200-/- controls and were therefore specific to the neuropathy. Furthermore, in S63del//PA200-/- mice, myelin thickness and nerve conduction were restored to WT levels. Thus, the upregulation of PA200 is maladaptive in S63del mice and its genetic ablation prevented neuropathy.

Gromwell ameliorates glucocorticoid-induced muscle atrophy through the regulation of Akt/mTOR pathway

BackgroundMuscle atrophy is characterized by decreased muscle mass, function, and strength. Synthetic glucocorticoids, including dexamethasone (Dexa), are commonly used to treat autoimmune diseases. However, prolonged exposure of Dexa with high dose exerts severe side effects, including muscle atrophy. The purpose of this study was to investigate whether Gromwell root extract (GW) can prevent Dexa-induced muscle atrophy in C2C12 cells and mice and to characterize the composition of GW to identify bioactive compounds.MethodsFor in vitro experiments, GW (0.5 and 1 µg/mL) or lithospermic acid (LA, 5 and 10 µM) was added to C2C12 myotubes on day 4 of differentiation and incubated for 24 h, along with 50 µM Dexa. For in vivo experiment, four-week-old male C57BL/6 mice were randomly divided into the four following groups (n = 7/group): Con group, Dexa group, GW0.1 group, and GW0.2 group. Mice were fed experimental diets of AIN-93 M with or without 0.1 or 0.2% GW for 4 weeks. Subsequently, muscle atrophy was induced by administering an intraperitoneal injection of Dexa at a dose of 15 mg/kg/day for 38 days, in conjunction with dietary intake.ResultsIn Dexa-induced myotube atrophy, treatment with GW increased myotube diameter, reduced the expression of muscle atrophy markers, and enhanced the expression of myosin heavy chain (MHC) isoforms in C2C12 cells. Supplementation with the GW improved muscle function and performance in mice with Dexa-induced muscle atrophy, evidenced in the grip strength and running tests. The GW group showed increased lean body mass, skeletal muscle mass, size, and myosin heavy chain isoform expression, along with reduced skeletal muscle atrophy markers in Dexa-injected mice. Supplementation with GW increased protein synthesis and decreased protein degradation through the Akt/mammalian target of rapamycin and glucocorticoid receptor/forkhead box O3 signaling pathways, respectively. We identified LA as a potential bioactive component of the GW. LA treatment increased myotube diameter and decreased the expression of muscle atrophy markers in Dexa-induced C2C12 cells.ConclusionsThese findings underscore the potential of the GW in preventing Dexa-induced skeletal muscle atrophy and highlight the contribution of LA to its effects.

The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery

Checkpoint activation after DNA damage causes a transient cell cycle arrest by suppressing cyclin-dependent kinases (CDKs). However, it remains largely elusive how cell cycle recovery is initiated after DNA damage. In this study, we discovered the upregulated protein level of MASTL kinase hours after DNA damage. MASTL promotes cell cycle progression by preventing PP2A/B55-catalyzed dephosphorylation of CDK substrates. DNA damage-induced MASTL upregulation was caused by decreased protein degradation, and was unique among mitotic kinases. We identified E6AP as the E3 ubiquitin ligase that mediated MASTL degradation. MASTL degradation was inhibited upon DNA damage as a result of the dissociation of E6AP from MASTL. E6AP depletion reduced DNA damage signaling, and promoted cell cycle recovery from the DNA damage checkpoint, in a MASTL-dependent manner. Furthermore, we found that E6AP was phosphorylated at Ser-218 by ATM after DNA damage and that this phosphorylation was required for its dissociation from MASTL, the stabilization of MASTL, and the timely recovery of cell cycle progression. Together, our data revealed that ATM/ATR-dependent signaling, while activating the DNA damage checkpoint, also initiates cell cycle recovery from the arrest. Consequently, this results in a timer-like mechanism that ensures the transient nature of the DNA damage checkpoint.

The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery.

Checkpoint activation after DNA damage causes a transient cell cycle arrest by suppressing cyclin-dependent kinases (CDKs). However, it remains largely elusive how cell cycle recovery is initiated after DNA damage. In this study, we discovered the upregulated protein level of MASTL kinase hours after DNA damage. MASTL promotes cell cycle progression by preventing PP2A/B55-catalyzed dephosphorylation of CDK substrates. DNA damage-induced MASTL upregulation was caused by decreased protein degradation, and was unique among mitotic kinases. We identified E6AP as the E3 ubiquitin ligase that mediated MASTL degradation. MASTL degradation was inhibited upon DNA damage as a result of the dissociation of E6AP from MASTL. E6AP depletion reduced DNA damage signaling, and promoted cell cycle recovery from the DNA damage checkpoint, in a MASTL-dependent manner. Furthermore, we found that E6AP was phosphorylated at Ser-218 by ATM after DNA damage and that this phosphorylation was required for its dissociation from MASTL, the stabilization of MASTL, and the timely recovery of cell cycle progression. Together, our data revealed that ATM/ATR-dependent signaling, while activating the DNA damage checkpoint, also initiates cell cycle recovery from the arrest. Consequently, this results in a timer-like mechanism that ensures the transient nature of the DNA damage checkpoint.

Azilsartan Ameliorates Skeletal Muscle Wasting in High Fat Diet (HFD)-induced Sarcopenic Obesity in Rats via Activating Akt Signalling Pathway

An association between the loss of skeletal muscle mass and obesity in the geriatric population has been identified as a disease known as sarcopenic obesity. Therefore, therapeutic/preventive interventions are needed to ameliorate sarcopenia. The present study investigates the effect of azilsartan (AZL) on skeletal muscle loss in High-Fat Diet (HFD)-induced sarcopenic obese (SO) rats. Four- and fourteen-months male Sprague Dawley rats were used and randomized in control and azilsartan treatment. 14 months animals were fed with HFD for four months and labeled as HFD-fed SO rats. Young & old rats received 0.5% carboxymethyl cellulose as a vehicle/AZL (8 mg/kg, per oral) treatment for six weeks. Grip strength and body composition analysis were performed after the last dose of AZL. Serum and gastrocnemius (GN)muscles were collected after animal sacrifice. AZL treatment significantly increased lean muscle mass, grip strength, myofibrillar protein, and antioxidant (superoxide dismutase & nitric oxide) levels in SO rats. AZL also restored the muscle biomarkers (creatine kinase, myostatin & testosterone), and insulin levels. AZL improves cellular, and ultracellular muscle structure and prevents type I to type II myofiber transitions in SO rats. Further, immunohistochemistry results showed increased expressions of pAkt and reduced expression of MuRF-1 and TNF-α exhibiting that AZL intervention could decrease protein degradation in SO rats. In conclusion, present results showed that AZL significantly increased lean mass, and restored muscle biomarkers, and muscle architecture. Taken together, the aforementioned findings suggest that azilsartan could be a possible therapeutic approach to reduce muscle wasting in sarcopenic obesity.

Therapeutic role of mTOR inhibitors in control of SARS-CoV-2 viral replication.

By the end of 2019, COVID-19 was reported in Wuhan city of China, and through human-human transmission, this virus spread worldwide and became a pandemic. Initial symptoms of the disease include fever, cough, loss of smell, taste, and shortness of breath, but a decrease in the oxygen levels in the body leads, and pneumonia may ultimately lead to the patient's death. However, the symptoms vary from patient to patient. To understand COVID-19 disease pathogenesis, researchers have tried to understand the cellular pathways that could be targeted to suppress viral replication. Thus, this article reviews the markers that could be targeted to inhibit viral replication by inhibiting the translational initiation complex/regulatory kinases and upregulating host autophagic flux that may lead to a reduction in the viral load. The article also highlights that mTOR inhibitors may act as potential inhibitors of viral replication. mTOR inhibitors such as metformin may inhibit the interaction of SARS-CoV-2 Nsp's and ORFs with mTORC1, LARP1, and 4E-BP. They may also increase autophagic flux by decreasing protein degradation via inhibition of Skp2, further promoting viral cell death. These events result in cell cycle arrest at G1 by p27, ultimately causing cell death.

Management and Treatment of Non-small Cell Lung Cancer with MET Alteration and Mechanisms of Resistance.

MET-driven tumors are a heterogenous group of non-small cell lung cancers (NSCLC) with activating mutations. Pathologic activation of MET can be achieved with increased number of gene copies overexpression, or decreased protein degradation through several mechanisms, including mutations, amplifications, or fusions. Besides its role as primary driver, MET activation might also mediate resistance to kinase inhibitors in NSCLC with various other actionable alterations. While checkpoint inhibitors have modest efficacy in MET-driven tumors, several approaches of targeted blockade are available. Among them the most promising are small tyrosine kinase inhibitors, antibody-drug conjugates, and bispecific antibodies. Unfortunately, resistance is virtually inevitable. Resistance to small kinase inhibitors might be mediated by kinase domain mutations or activation of shunting cascades. Various resistance mechanisms might be present in one patient, making it overcoming an unresolved problem.

Effect of ultra-high pressure on the relationship between endogenous proteases and protein degradation of Yesso scallop (Mizuhopecten yessoensis) adductor muscle during iced storage.

This study aimed to explore the effect of ultra-high pressure (UHP) treatment (100-500MPa, 5min, 15±1℃) on the relationship between endogenous proteases and protein degradation of Yesso scallop (Mizuhopecten yessoensis) adductor muscle during iced storage for 28days. Our findings showed that the UHP treatment kept the water holding capacity stable, increased the hardness and decreased the springiness of scallop adductor muscle during iced storage. 400 and 500MPa UHP treatments caused protein denaturation and oxidation significantly, decreased protein degradation rate and inhibited the activities of endogenous proteases. According to the correlation analysis, the activities of cathepsin B, D, H, L, calpain and serine protease were positively correlated with TCA-soluble peptides. The activities of endogenous proteases were significantly correlated with protein degradation. Therefore, the effect of UHP on endogenous protease caused the protein degradation rate to slow down and prevented the texture deterioration in scallops.

Leucine Supplementation in Cancer Cachexia: Mechanisms and a Review of the Pre-Clinical Literature.

Cancer cachexia (CC) is a complex syndrome of bodily wasting and progressive functional decline. Unlike starvation, cachexia cannot be reversed by increased energy intake alone. Nonetheless, targeted nutritional support is a necessary component in multimodal syndrome management. Due to the highly catabolic nature of cancer cachexia, amino acid supplementation has been proposed. Interestingly, leucine has been found to increase protein synthesis and decrease protein degradation via mTORC1 pathway activation. Multiple pre-clinical studies have explored the impact of leucine supplementation in cachectic tumor-bearing hosts. Here, we provide an overview of leucine’s proposed modes of action to preserve lean mass in cachexia and review the current pre-clinical literature related to leucine supplementation during CC. Current research indicates that a leucine-rich diet may attenuate CC symptomology; however, these works are difficult to compare due to methodological differences. There is need for further pre-clinical work exploring leucine’s potential ability to modulate protein turnover and immune response during CC, as well as the impact of additive leucine on tumor growth.

Megasphaera elsdenii and Saccharomyces Cerevisiae as direct fed microbials during an in vitro acute ruminal acidosis challenge

This study aimed to evaluate the effects of Saccharomyces cerevisiae and Megasphaera elsdenii as direct fed microbials (DFM) in beef cattle finishing diets to alleviate acute ruminal lactic acidosis in vitro. A dual-flow continuous culture system was used. Treatments were a Control, no DFM; YM1, S. cerevisiae and M. elsdenii strain 1; YM2, S. cerevisiae and M. elsdenii strain 2; and YMM, S. cerevisiae and half of the doses of M. elsdenii strain 1 and strain 2. Each DFM dose had a concentration of 1 × 108 CFU/mL. Four experimental periods lasted 11 days each. For the non-acidotic days (day 1–8), diet contained 50:50 forage to concentrate ratio. For the challenge days (day 9–11), diet contained 10:90 forage to concentrate ratio. Acute ruminal acidosis was successfully established. No differences in pH, d-, l-, or total lactate were observed among treatments. Propionic acid increased in treatments containing DFM. For N metabolism, the YMM treatment decreased protein degradation and microbial protein synthesis. No treatment effects were observed on NH3–N concentration; however, efficiency of N utilization by ruminal bacteria was greater than 80% during the challenge period and NH3–N concentration was reduced to approximately 2 mg/dL as the challenge progressed.

Ubiquitylation of unphosphorylated c-myc by novel E3 ligase SCFFbxl8

ABSTRACT Overexpression of c-myc via increased transcription or decreased protein degradation is common to many cancer etiologies. c-myc protein degradation is mediated by ubiquitin-dependent degradation, and this ubiquitylation is regulated by several E3 ligases. The primary regulator is Fbxw7, which binds to a phospho-degron within c-myc. Here, we identify a new E3 ligase for c-myc, Fbxl8 (F-box and Leucine Rich Repeat Protein 8), as an adaptor component of the SCF (Skp1-Cullin1-F-box protein) ubiquitin ligase complex, for selective c-myc degradation. SCFFbxl8 binds and ubiquitylates c-myc, independent of phosphorylation, revealing that it regulates a pool of c-myc distinct from SCFFbxw7. Loss of Fbxl8 increases c-myc protein levels, protein stability, and cell division, while overexpression of Fbxl8 reduces c-myc protein levels. Concurrent loss of Fbxl8 and Fbxw7 triggers a robust increase in c-myc protein levels consistent with targeting distinct pools of c-myc. This work highlights new mechanisms regulating c-myc degradation.

Schisandrae chinensis Fructus Extract Ameliorates Muscle Atrophy in Streptozotocin-Induced Diabetic Mice by Downregulation of the CREB-KLF15 and Autophagy-Lysosomal Pathways.

Type 1 diabetes mellitus is an autoimmune disease caused by the destruction of pancreatic beta cells. Many patients with type 1 diabetes experience skeletal muscle wasting. Although the link between type 1 diabetes and muscle wasting is not clearly known, insulin insufficiency and hyperglycemia may contribute to decreased muscle mass. In this study, we investigated the therapeutic effect of the ethanolic extract of Schisandrae chinensis Fructus (SFe) on muscle wasting in streptozotocin (STZ)-induced diabetic mice. STZ-diabetic C57BL/6 mice (blood glucose level ≥300 mg/dL) were orally administered SFe (250 or 500 mg/kg/day) for 6 weeks. We observed that SFe administration did not change blood glucose levels but increased gastrocnemius muscle weight, cross-sectional area, and grip strength in STZ-induced diabetic mice. Administration of SFe (500 mg/kg) decreased the expression of atrophic factors, such as MuRF1 and atrogin-1, but did not alter the expression of muscle synthetic factors. Further studies showed that SFe administration decreased the expression of KLF15 and p-CREB, which are upstream molecules of atrophic factors. Examination of the expression of molecules involved in autophagy–lysosomal pathways (e.g., p62/SQSTM1, Atg7, Beclin-1, ULK-1, LC3-I, and LC3-II) revealed that SFe administration significantly decreased the expression of p62/SQSTM1, LC3-I, and LC3-II; however, no changes were observed in the expression of Atg7, Beclin-1, or ULK-1. Our results suggest that SFe ameliorated muscle wasting in STZ-induced diabetic mice by decreasing protein degradation via downregulation of the CREB-KLF15-mediated UPS system and the p62/SQSTM1-mediated autophagy–lysosomal pathway.

Administration of tauroursodeoxycholic acid attenuates dexamethasone-induced skeletal muscle atrophy

Glucocorticoids are known to induce skeletal muscle atrophy by suppressing protein synthesis and promoting protein degradation. Tauroursodeoxycholic acid (TUDCA) has beneficial effects in several diseases, such as hepatobiliary disorders, hindlimb ischemia and glucocorticoid-induced osteoporosis. However, the effects of TUDCA on glucocorticoid -induced skeletal muscle atrophy remains unknown. Therefore, in the present research, we explored the effects of TUDCA on dexamethasone (DEX)-induced loss and the potential mechanisms involved. We found TUDCA alleviated DEX-induced muscle wasting in C2C12 myotubes, identified by improved myotube differentiation index and expression of myogenin and MHC. And it showed that TUDCA activated the Akt/mTOR/S6K signaling pathway and inhibited FoxO3a transcriptional activity to decreased expression of MuRF1 and Atrogin-1, while blocking Akt by MK2206 blocked these effects of TUDCA on myotubes. Besides, TUDCA also attenuated DEX-induced apoptosis of myotubes. Furthermore, TUDCA was administrated to the mouse model of DEX-induced skeletal muscle atrophy. The results showed that TUDCA improved DEX-induced skeletal muscle atrophy and weakness (identified by increased grip strength and prolonged running exhaustive time) in mice by suppression of apoptosis, reduction of protein degradation and promotion of protein synthesis. Taken together, our research proved for the first time that TUDCA protected against DEX-induced skeletal muscle atrophy not only by improving myogenic differentiation and protein synthesis, but also through decreasing protein degradation and apoptosis of skeletal muscle.

Abstract 1962: Voruciclib, a CDK9 inhibitor, downregulates MYC and inhibits proliferation of KRAS mutant cancers in preclinical models

Abstract Mutations in KRAS at G12, G13, and Q61 are oncogenic drivers in many cancers, including lung, colorectal, pancreatic, multiple myeloma, and uterine carcinomas. KRAS mutations are frequently accompanied by stabilization of the MYC oncoprotein through increased MYC transcription and decreased protein degradation that is mediated by phosphorylation of MYC on Ser 62 by ERK and CDK9 kinases. Voruciclib is a novel oral inhibitor of CDKs 9, 4, 6, and 1 that is currently being tested in Phase 1B clinical trials for B-cell malignancies and acute myeloid leukemia. Voruciclib inhibition of CDK9 leads to decreased expression of transcriptional targets of RNA Pol II, such as Mcl1 and MYC. To test whether voruciclib could be effective in cancers driven by dysregulated KRAS-MYC signaling, ~20 cancer cell lines with KRAS mutations (G12A, G12C, G12D, G12S, G12V, G13C, G13D, Q61H) were treated in preclinical studies with voruciclib. MTS and Cell Titer Glo assays were used to monitor growth in vitro. Voruciclib decreased viability in all cell lines tested. To investigate MYC protein stability, MIA PACA-2 (G12C) cells were treated with 4 µM voruciclib for 5-240 min, followed by SDS-PAGE and Western Blotting analysis with α-MYC and α-pSer62-MYC antibodies. Voruciclib treatment resulted in a reduction in phosphorylation of MYC on Ser 62. A 60% decrease in pSer62 was observed after 5 min that reached 80% by 60 min. In contrast, there was no decrease in total MYC protein at either 5 or 15 min. A 10% reduction in total MYC was observed at 60 min that reached 50% at 240 min. The ability of voruciclib to inhibit tumor growth in vivo was also tested in murine xenograft models. KRAS mutant human cancer cells HCT-116 (CRC, KRAS G13D), SW-480 (CRC, KRAS G12V), and H-460 (NSCLC, KRAS Q61H) were injected subcutaneously into SCID mice. Once tumors reached 5-10 mm in diameter, voruciclib or vehicle were administered orally at 50, 100, or 200 mg/kg OD for 11-14 days. Tumors were measured every 2-3 days, and growth inhibition relative to control was calculated. Significant tumor growth inhibition (>50%) was observed at all doses of voruciclib tested. Collectively, these data demonstrate that voruciclib inhibition of CDK9 leads to reduced phosphorylation of MYC on Ser 62 followed by a decrease in total MYC protein in MIA PACA-2 cells and inhibition of growth in multiple KRAS mutant cancer cell lines in vivo and in vitro. This suggests that voruciclib could be an attractive therapeutic option for cancers driven by KRAS-MYC. Citation Format: Sandra E. Wiley, Yongwei Su, Yubin Ge. Voruciclib, a CDK9 inhibitor, downregulates MYC and inhibits proliferation of KRAS mutant cancers in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1962.

Torpor versus interbout arousal: what is more important for protein metabolism and regeneration?

Hibernation is used by specie as diverse as ground squirrels, snakes and bats to cope with reduced food availability and lowered temperatures and is a unique model for studying the molecular mechanisms of plasticity in striated muscles. Important questions in modern muscle biology are why some skeletal muscles atrophy and other muscles such as the diaphragm and the heart hypertrophy during hibernation, and why, despite the atrophy, skeletal muscle strength and fatigue resistance are maintained after arousal. The molecular mechanisms responsible are of interest both from a fundamental biological viewpoint and as a potential therapy for patients in critical care units. In this issue of Experimental Physiology, Yan et al. (2021) shed light on the distinctive features of the molecular mechanisms involved in the development of the hypertrophic response in the diaphragm and the atrophic response in the gastrocnemius muscle in hibernating Daurian ground squirrels. In particular, the authors showed that a decrease in protein synthesis, an increase in protein degradation and a decrease in muscle regeneration potential contributed to the atrophy of the gastrocnemius muscles during torpor, while an increase in protein synthesis, a decrease in protein degradation and an increase in muscle regeneration potential contributed to diaphragm muscle hypertrophy. In discussing these new and interesting results, it can be assumed, however, that a significant contribution to the formation of hypertrophic or atrophic muscle phenotype could have been made by changes in anabolic and catabolic processes occurring in interbout arousals, as indicated by the results of recent studies (Chang et al., 2020; Popova et al., 2020 ). Further research is needed to show how important interbout arousals are in the regulation of protein turnover and regeneration in different striated muscles in hibernating animals. Understanding these and other molecular mechanisms is important for choosing strategies and methods for treatment of skeletal muscle pathologies and diseases of the cardiovascular system, as well as for avoiding injurious interventions in cases of energy deficits in critical illness (Stanzani et al., 2020). None declared.

Differential protein metabolism and regeneration in hypertrophic diaphragm and atrophic gastrocnemius muscles in hibernating Daurian ground squirrels.

What is the central question of this study? The aim was to investigate whether diaphragm hypertrophy and gastrocnemius atrophy during hibernation of Daurian ground squirrels involve differential regulation of protein metabolism and regeneration. What is the main finding and its importance? We clarified the differences in protein metabolism and muscle regenerative potential in the diaphragm and gastrocnemius of hibernating ground squirrels, reflecting the different adaptability of muscles. Are differences in the regulation of protein metabolism and regeneration involved in the different phenotypic adaptation mechanisms of muscle hypertrophy and atrophy in hibernators? Two fast-type muscles (diaphragm and gastrocnemius) in summer active and hibernating Daurian ground squirrels were selected to detect changes in cross-sectional area (CSA) and protein expression indicative of protein synthesis metabolism (protein expression of P-Akt, P-mTORC1, P-S6K1 and P-4E-BP1), protein degradation metabolism (MuRF1, atrogin-1, calpain-1, calpain-2, calpastatin, desmin, troponinT, Beclin1 and LC3-II) and muscle regeneration (MyoD, myogenin and myostatin). In the hibernation group compared with the summer active group, the CSA of the diaphragm muscle increased significantly by 26.1%, whereas the CSA of the gastrocnemius muscle decreased significantly by 20.4%. Our study also indicated that increased protein synthesis, decreased protein degradation and increased muscle regenerative potential contributed to diaphragm muscle hypertrophy, whereas decreased protein synthesis, increased protein degradation and decreased muscle regenerative potential contributed to gastrocnemius muscle atrophy. In conclusion, the differences in muscle regeneration and regulatory pattern of protein metabolism might contribute to the different adaptive changes observed in the diaphragm and gastrocnemius muscles of ground squirrels.

Conserving purple prairie clover (Dalea purpurea Vent.) as hay and silage had little effect on the efficacy of condensed tannins in modulating ruminal fermentation in vitro.

The objective of this study was to determine the effects of forage conservation method and condensed tannins (CT) in conserved forage on rumen fermentation. Purple prairie clover (PPC; Dalea purpurea Vent.) containing 84.5 g kg-1 dry matter (DM) of CT was harvested at full flower and preserved as freeze-dried green chop (F), hay (H) or silage (S). Batch culture incubations were conducted using conserved forages as a substrate and treatments were arranged as 3 × 2 factorial design of forage type (F, H and S) with or without polyethylene glycol (PEG). PEG was used to isolate the effects of CT on fermentation. Incubation was repeated twice with quadruplicate vials for each treatment in each incubation. 15 N-labelled ammonium sulfate was used as microbial N marker and headspace gas was sampled to determine methane production. Concentrations of neutral detergent fiber (NDF) and acid detergent fiber were lower (P < 0.01) in F than in H or S. Ensiling decreased (P < 0.001) total phenolics and extractable CT, but had no effect on total CT, whereas none of these phenolic fractions were altered in H. Hay and silage had lower (P < 0.01) true DM disappearance (TDMD) and NDF disappearance (NDFD) than F. Inclusion of PEG did not affect TDMD or NDFD after 8, 24 or 72 h of incubation. Productions of total gas, methane and total volatile fatty acid and the rate of gas production (c) were also similar, but ammonia was higher (P < 0.05) and microbial N was lower (P < 0.05) with than without PEG after 72 h of incubation. After 72 h, a lower (P < 0.001) proportion of acetate but higher (P < 0.05) proportion of propionate was noted with S, resulting in a lower (P < 0.001) acetate:propionate ratio as compared to F or H after 8 h of incubation. Condensed tannins in PPC decreased protein degradation in vitro, but had minimal effects on overall rumen fermentation, and conservation of PPC as hay or silage had little effect on the efficacy of CT in modulating rumen fermentation. © 2020 Her Majesty the Queen in Right of Canada. Journal of The Science of Food and Agriculture © 2020 Society of Chemical Industry.

Mitigating the Astringency of Acidified Whey Protein in Proteinaceous High Internal Phase Emulsions.

The demand for high-protein food products has rapidly increased in recent years because of the growing health awareness of consumers. Whey protein is an abundant byproduct from the dairy industry with a high nutritional value. However, it is a substantial challenge to mitigate the astringent taste in acidified whey protein food products. Water-in-oil (W/O) emulsion with a high internal aqueous phase represents a unique opportunity to simultaneously deliver the high concentration of whey protein and mitigate the astringent taste in spreadable foods. To incorporate protein into the high internal phase emulsion (HIPE), whey protein-polysaccharide complexes (PPCs) are formed to minimize destabilization of the emulsion. Such whey PPCs are found to not only stabilize protein but also play a role in mitigating astringency. The protein astringency is studied through real-time monitoring of the protein-saliva mucin interaction on a quartz crystal microbalance. Our results showed that PPC showed less mucin interaction compared to whey protein and preventing protein-mucin interaction can lead to decreased astringency perception. By fabricating the PPC into the HIPE, the astringency of protein can be further minimized with improved protein loading capacity (20 wt %) and viscoelasticity (103-104 Pa). Incorporation of whey protein at acidic pH in W/O HIPE imparts advantages, such as decreased protein degradation as well as increased emulsion spreadability and stability. Furthermore, the resultant emulsions are less astringent in sensory studies, indicating HIPE's potential to integrate acidified whey protein at a high concentration with a low astringency profile.

Effect of dietary inclusion of guanidinoacetic acid on broiler performance

Background: Creatine and its precursor, guanidinoacetic acid (GAA), decrease protein degradation, avoiding amino acid offset to gluconeogenesis. Objective: To evaluate the inclusion of GAA in the pre-initial diet (1 to 7 days) on performance, digestibility, and blood parameters of broilers from one to 21 days of age. Methods: 252 male broiler chickens aged one day were distributed in a completely randomized design with seven replicates and 12 animals per experimental unit. Treatments consisted of a basal diet, and the basal added with 0.10 and 0.20% GAA. Results: From days 1 to 7, a tendency on improved feed conversion (FC) was observed for broilers receiving 0.20% GAA (p&lt;0.10). Between 7 and 14 days, FC improved, as well as a tendency was observed on improved weight gain in animals fed 0.20% GAA. Animals that received 0.20% GAA showed higher blood concentrations of creatine kinase. Blood creatine and blood glucose did not differ between treatments at day six. The assimilation coefficient of crude protein increased during days 3 to 7 for animals receiving 0.20% GAA. On the period between 17 and 21 days, no difference was observed between treatments. Intestinal weight and length did not show significant differences. Conclusion: Dietary inclusion of 0.20% GAA during the pre-initial stage could improve weight gain and feed conversion of broilers between 1 and 14 days of age.

Label-free based proteomics analysis of protein changes in frozen whiteleg shrimp (Litopenaeus vannamei) pre-soaked with sodium trimetaphosphate

Muscle proteins in peeled shrimp (Litopenaeus vannamei) are known to be unstable and prone to denaturation affected by freezing and frozen storage. In this study, label-free proteomics were performed to explore the stabilization of frozen (30 days at −18 °C) shrimp muscle proteins when a pre-soaking treatment with distilled water (DW)- or sodium trimetaphosphate (ST) was applied; comparison to fresh samples (FS) was carried out. In total, 163 differentially abundant proteins (DAPs) were down-regulated in DW batch when compared to FS, these including ribosomal proteins, actins, myosin, paramyosin, myosin heavy chains, and tropomyosin; interestingly, most of these DAPs (181 proteins) were up-regulated in ST batch when compared to DW shrimp, mainly due to the incorporation of ST into muscle tissues. The results revealed the decreased protein degradation resulting from the reduced damage from ice-crystal growth. Gene ontology (GO) analysis suggested that these DAPs were mainly involved in catalytic activity, binding, and metabolic processes. Kyoto encyclopedia of genes and genomes (KEGG) results indicated that many pathways, including phototransduction, metabolic, and ribosomal pathways that interacted with phosphoglycerate mutase, actins, and ribosomal proteins were altered. Additionally, Eukaryotic clusters of orthologous group (KOG) results confirmed that incorporated ST maintained the stability of these DAPs in shrimp muscle, especially for cytoskeleton proteins, and retarded the degradation of muscle proteins during frozen storage.