Ursodeoxycholic acid induces sarcopenia associated with decreased protein synthesis and autophagic flux

Background Skeletal muscle generates force and movements and maintains posture. Under pathological conditions, muscle fibers suffer an imbalance in protein synthesis/degradation. This event causes muscle mass loss and decreased strength and muscle function, a syndrome known as sarcopenia. Recently, our laboratory described secondary sarcopenia in a chronic cholestatic liver disease (CCLD) mouse model. Interestingly, the administration of ursodeoxycholic acid (UDCA), a hydrophilic bile acid, is an effective therapy for cholestatic hepatic alterations. However, the effect of UDCA on skeletal muscle mass and functionality has never been evaluated, nor the possible involved mechanisms. Methods We assessed the ability of UDCA to generate sarcopenia in C57BL6 mice and develop a sarcopenic-like phenotype in C2C12 myotubes and isolated muscle fibers. In mice, we measured muscle strength by a grip strength test, muscle mass by bioimpedance and mass for specific muscles, and physical function by a treadmill test. We also detected the fiber's diameter and content of sarcomeric proteins. In C2C12 myotubes and/or isolated muscle fibers, we determined the diameter and troponin I level to validate the cellular effect. Moreover, to evaluate possible mechanisms, we detected puromycin incorporation, p70S6K, and 4EBP1 to evaluate protein synthesis and ULK1, LC3 I, and II protein levels to determine autophagic flux. The mitophagosome-like structures were detected by transmission electron microscopy. Results UDCA induced sarcopenia in healthy mice, evidenced by decreased strength, muscle mass, and physical function, with a decline in the fiber's diameter and the troponin I protein levels. In the C2C12 myotubes, we observed that UDCA caused a reduction in the diameter and content of MHC, troponin I, puromycin incorporation, and phosphorylated forms of p70S6K and 4EBP1. Further, we detected increased levels of phosphorylated ULK1, the LC3II/LC3I ratio, and the number of mitophagosome-like structures. These data suggest that UDCA induces a sarcopenic-like phenotype with decreased protein synthesis and autophagic flux. Conclusions Our results indicate that UDCA induces sarcopenia in mice and sarcopenic-like features in C2C12 myotubes and/or isolated muscle fibers concomitantly with decreased protein synthesis and alterations in autophagic flux.

Guanidinoacetic acid supplementation on growth performance and molecular mechanisms of lean mass gain in nursery pigs / Suplementação de ácido guanidinoacético no desempenho e mecanismo moleculares de ganho de massa magra em leitões

ABSTRACT: The objective with the present study was to evaluate the effect of guanidinoacetic acid (GAA) on the growth performance of nursery piglets as well as a possible molecular mechanism of action on lean mass gain. Seventy-two pigs, weaned at 21 d, weighing 6.80 ± 1.2 kg were distributed in a completely randomized design into one of three dietary treatments (control, control + 1.2 g/kg GAA or control + 2.4 g/kg GAA) and 8 replicates per treatment. The control diet was an animal protein-free diet based on corn and soybean meal. Body weight, average daily weight gain, average daily feed intake and feed efficiency were evaluated at 35, 49, and 56 days. At the end of the experiment, one animal per pen was slaughtered and samples of the vastus lateralis muscle were collected for RT-qPCR and protein abundance analysis. Overall (from 21 to 56 d), GAA supplementation improved feed efficiency (P < 0.03). Skeletal muscle of pigs fed with GAA diet had greater mRNA expression of Akt (P < 0.04) and RPS6KB2 (P<0.01). In conclusion, supplementation with 2.4 g/kg GAA to nursery piglets improves feed efficiency and activates molecular mechanisms important to lean mass gain.

RESUMO: O objetivo do presente estudo foi avaliar o efeito do ácido guanidinoacético (GAA) no desempenho de leitões, bem como um possível mecanismo de ação molecular no ganho de massa magra. Setenta e dois leitões, desmamados aos 21 dias, pesando 6,80 ± 1,2 kg, foram distribuídos em um delineamento inteiramente casualizado com três tratamentos dietéticos (controle, controle + 1,2 g / kg ou controle + 2,4 g / kg GAA) e 8 repetições por tratamento. A dieta controle não continha proteína animal e foi formulada a base de milho e farelo de soja. O desempenho dos animais foi avaliado aos 35, 49 e 56 dias. Ao final do experimento, um animal por unidade experimental foi abatido e amostras do músculo Vastus lateralis foram coletadas para análise de RT-qPCR e abundância de proteínas. A suplementação com GAA melhorou a eficiência alimentar (P<0,03) aos 56 dias. O músculo dos leitões suplementados apresentou maior expressão de mRNA de Akt (P<0,04) e RPS6KB2 (P <0,01). Em conclusão, a suplementação de 2,4 g / kg de GAA em leitões (21 a 56 d) melhora a eficiência alimentar e ativa mecanismos moleculares importantes para o ganho de massa magra.

Acacia mearnsii tannin extract as a feed additive: impact on feed intake, digestibility and nitrogen excretion by sheep fed a tropical grass-based diet / Extrato tanífero de Acacia mearnsii como aditivo alimentar: impacto sobre o consumo, digestibilidade e excreção de nitrogênio em ovinos alimentados com uma dieta a base de gramínea tropical

ABSTRACT: The aim of this study was to evaluate the effect of the dietary inclusion of Acacia mearnsii tannin extract (TA) on nutrients intake and digestibility, and nitrogen (N) retention by sheep given a tropical grass-based diet. The trial was conducted with six castrated male sheep in a cross-over design in two 21-days experimental periods. The sheep were housed in metabolic cages and offered Tifton 85 hay (Cynodon dactylon) ad libitum plus concentrate at a rate of 12 g of dry matter (DM)/kg body weight (BW). The treatments were concentrate without (Control) or with 10 g TA/kg DM (Tannin). Concentration of TA in the diet was 3.8 g/kg DM and did not affect the feed intake or apparent digestibility. The TA decreased the true digestibility of n compounds (P<0.05) whereas did not impact the N retention, microbial N flow to the small intestine or the efficiency of rumen microbial protein synthesis. In conclusion, a low dietary concentration of TA did not impact the nutrients supply and N use by sheep fed with a tropical grass-based diet.

RESUMO: O objetivo do presente estudo foi avaliar o efeito da inclusão alimentar de extrato tanífero de Acacia mearnsii (TA) sobre o consumo, a digestibilidade e a retenção de nitrogênio (N) em ovinos alimentados com uma dieta a base de gramínea tropical. O experimento foi conduzido com seis ovinos machos castrados, em delineamento em reversão simples, com dois períodos experimentais de 21 dias cada. Os animais foram alojados em gaiolas metabólicas e alimentadas ad libitum com feno de Tifton 85 (Cynodon dactylon) mais concentrado oferecido a uma taxa de 12 g de matéria seca (MS)/kg de peso corporal. Os tratamentos foram: concentrado sem (Controle) ou com 10 g de TA/kg MS (Tanino). A concentração de TA na dieta foi de 3,8 g/kg MS e não afetou o consumo e nem a digestibilidade aparente dos nutrientes. O TA diminuiu a digestibilidade verdadeira do N (P<0,05), mas não afetou a retenção de N, o fluxo microbiano de N para o intestino delgado ou a eficiência de síntese de proteína microbiana no rúmen. Em conclusão, a inclusão de uma baixa dose de TA/kg MS na dieta não afetou a oferta de nutrientes nem o uso de N em ovinos alimentados com uma dieta baseada em uma gramínea tropical.

L-asparaginase de Erwinia chrysanthemi: expressão proteica livre de células e bioconjugação a bacteriófagos / L-asparaginase from Erwinia chrysanthemi: cell-free protein expression and bioconjugation to bacteriophages

A L-Asparaginase (L-ASNase) de Erwinia chrysathemi (ErA) é uma enzima amplamente utilizada para o tratamento da leucemia linfoblástica aguda (LLA). Embora o seu uso como segunda linha de tratamento para a LLA tenha proporcionado consideráveis benefícios clínicos, reações de hipersensibilidade e rápida depuração plasmática ainda são problemas recorrentes. Ademais, extensivos e custosos processos de produção da ErA são necessários para a obtenção da enzima pura. Com base nesses problemas, o presente trabalho propõe (1) o estudo de viabilidade de expressão da ErA em um sistema de síntese proteica livre de células (SPLC) e (2) a conjugação da proteína em bacteriófagos como ferramenta alternativa para o isolamento e monitoramento da depuração plasmática da ErA. Foram utilizados extratos celulares de Escherichia coli suplementados com solução energética contendo creatina fosfato (CP) como fonte de energia para síntese in vitro de ErA. Para conjugação da ErA a bacteriófagos, o sistema SpyTag/SpyCatcher foi implementado: SpyCatcher foi fusionado à porção N-terminal da ErA e bacteriófagos filamentosos da linhagem M13 e fd foram modificados de modo a expressar SpyTag nas proteínas de capsídeo pIII e pVIII, respectivamente. Em relação ao primeiro objetivo, o sistema de SPLC foi capaz de expressar a ErA com atividade. A proteína foi expressa na fração solúvel e apresentou atividade enzimática significativamente superior em relação à reação controle (7,07 ± 0,68 U/mL vs. 1,83 ± 0,14 U/mL). Tempo necessário para obtenção do extrato celular foi reduzido de 45 para 26 hrs, e sete componentes da solução energética foram removidos da composição original sem implicações negativas na eficiência de expressão da ErA, simplificando desta forma o processo de SPLC. Em relação ao segundo objetivo, ErA fusionada à SpyCatcher (SpyCatcher_ErA) foi conjugada com êxito em bacteriófagos capazes de expressar SpyTag fusionadas na porção N-terminal das proteínas pIII (SpyTag_pIII) e pVIII (SpyTag_pVIII). A porcentagem de formação dos conjugados entre SpyCatcher_ErA e SpyTag_pIII ((ErA)5-pIII) foi de 6% enquanto formação dos conjugados entre SpyCatcher_ErA e SpyTag_pVIII ((ErA)50-pVIII) foi de 46%, valores estes confirmados por atividade enzimática. Solução contendo conjugados foram injetados em camundongos e sequenciados/titulados com êxito. Não houve diferença de depuração plasmática entre (ErA)5-pIII e bacteriófago controle, mas houve maior taxa de eliminação de (ErA)50-pVIII em relação ao mesmo bacteriófago não conjugado à SpyCatcher_ErA. Os resultados aqui apresentados confirmam ser possível expressar ErA com atividade biológica em sistemas de SPLC. Além disso, o sistema de conjugação da ErA a bacteriófagos aqui desenvolvido foi capaz de monitorar a concentração de ErA presente na circulação em função do tempo, tornando-se uma potencial plataforma de desenvolvimento de novas proteoformas da ErA com características clínicas melhoradas

L-Asparaginase (L-ASNase) from Erwinia chrysanthemi (ErA) is a widely used enzyme for treatment of acute lymphoblastic leukemia (ALL). Although its use as a second-line treatment has provided significant clinical benefits, hypersensitivity reactions and a fast clearance rate are recurring L-ASNase-related problems. In addition, extensive and costly production processes are required for the manufacturing of pure ErA. Based on these drawbacks, this current work proposes (1) the study of the use of a cell-free protein synthesis (CFPS) system as a viable platform for the synthesis of ErA and (2) the conjugation of the protein on bacteriophages as an alternative tool for the isolation and monitoring of ErA clearance. Escherichia coli-derived cell extracts supplemented with a creatine phosphate-based energy solution were used to synthesize ErA in vitro. To conjugate ErA on bacteriophages, the SpyTag/SpyCatcher system was implemented: SpyCatcher was fused to the N-terminus of the ErA while filamentous phage strains M13 and fd were engineered in order to display SpyTag on their pIII and pVIII capsid proteins, respectively. Regarding the first goal, the CFPS system was able to express an active ErA. The protein was expressed in the soluble fraction and there presented a significant higher enzymatic activity compared to the control reaction (7.07 ± 0.68 U/mL vs. 1.83 ± 0.14 U/mL). Time required to obtain the cell extract was reduced from 45 to 26 hours, and seven energy solution reagents were removed from the original solution without compromising the efficiency of ErA expression, thus simplifying the CFPS process. With respect to the second goal, ErA fused to SpyCatcher (SpyCatcher_ErA) was sucessfully conjugated on bacteriophages capable of displaying SpyTag fused to the Nterminus of the pIII (SpyTag_pIII) or pVIII (SpyTag_pVIII) proteins. Percentage of conjugate formation between SpyCatcher_ErA and SpyTag_pIII (ErA)5-pIII was 6% whereas conjugate formation between SpyCatcher_ErA and SpyTag_pVIII (ErA)50-pVIII was 46%, values that were confirmed by enzymatic activity. Sample containing conjugates were injected into mice and sucessfully sequenced/titrated. No clearance differences were observed between (ErA)5- pIII and a control bacteriophage, but a higher clearance rate was observed for (ErA)50-pVIII compared to SpyTag_VIII non conjugated to SpyCatcher_ErA. The results here presented confirm the expression of a biologically active ErA from a CFPS system. Besides, the development of a conjugation system capable of linking ErA to bacteriophages could be used as a means to monitor the ErA concentration in the blood as a function of time and also as a potential platform to be used in the development of novel ErA proteoforms with improved clinical properties

Mechanism of ribosome biogenesis and exercise adaptation of skeletal muscle hypertrophy / 中国组织工程研究

BACKGROUND: Research evidence shows that skeletal muscle contractile activity can induce ribosomal biogenesis, which plays an important role in the control of skeletal muscle mass. OBJECTIVE: To review the main mechanism of ribosome biogenesis in skeletal muscle hypertrophy, upstream regulatory signals of ribosomal biogenesis in skeletal muscle, and effect of exercise on ribosomal biogenesis, and to explore the ribosome biogenesis mechanism of exercise-induced skeletal muscle hypertrophy. METHODS: Relevant studies about exercise, skeletal muscle hypertrophy and ribosome biogenesis in CNKI, Wanfang, and PubMed databases were searched. The key words were “exercise, resistance training, skeletal muscle hypertrophy, protein synthesis, ribosome biogenesis” in English and Chinese. Relevant literatures published from 1999 to 2019 were searched and screened according to inclusion and exclusion criteria. RESULTS AND CONCLUSION: (1) Ribosome biogenesis as a main source of translational capacity plays an important role in muscle growth. (2) A single bout of resistance exercise can promote the ribosome biogenesis. However, cumulative bouts of resistance exercise eventually lead to the accumulation of mature rRNAs, leading to increased concentration of total RNA, which promote the growth of skeletal muscle. (3) Ribosome biogenesis may be the key molecular mechanism for the regulation of skeletal muscle hypertrophy induced by resistance training. (4) Moderate-volume resistance training led to adaptations to resistance training. This hypertrophy was associated with volume-dependent regulation of total RNA. This suggests that ribosomal biogenesis regulates the dose-effect relationship between training volume and muscle hypertrophy.

Short-term creatine supplementation changes protein metabolism signaling in hindlimb suspension

The effect of a short-term creatine supplementation on hindlimb suspension (HS)-induced muscle atrophy was investigated. Creatine monohydrate (5 g/kg b.w. per day) or placebo, divided in 2 daily doses, was given by oral gavage for 5 days. Rats were maintained in HS with dietary supplementation concomitantly for 5 days. Body weight, soleus and EDL muscle masses, and cross-sectional areas (CSA) of the muscle fibers were measured. Signaling pathways associated with skeletal muscle mass regulation (FST, MSTN, FAK, IGF-1, MGF, Akt, mTOR, atrogin-1, and MuRF1 expressions, and Akt, S6, GSK3B, and 4EBP1 proteins) were evaluated in the muscles. Soleus muscle exhibited more atrophy than the EDL muscle due to HS. Creatine supplementation attenuated the decrease of wet weight and increased p-4EBP1 protein in the EDL muscle of HS rats. Also, creatine increased mTOR and atrogin-1 expressions in the same muscle and condition. In the absence of HS, creatine supplementation increased FAK and decreased MGF expressions in the EDL muscle. Creatine attenuated the increase in FST expression due to HS in the soleus muscle. MuRF1 expression increased in the soleus muscle due to creatine supplementation in HS animals whereas atrogin-1 expression increased still further in this group compared with untreated HS rats. In conclusion, short-term creatine supplementation changed protein metabolism signaling in soleus and EDL muscles. However, creatine supplementation only slightly attenuated the mass loss of both muscles and did not prevent the CSA reduction and muscle strength decrease induced by HS for 5 days.

Effects of Electroacupuncture on Mammalian Target of Rapamycin/70-KD Ribosomal Protein S6 Kinase Pathway in Gastrocnemius Muscle in Denervated Rats / 中国康复理论与实践

@#Objective To explore the effects and mechanism of electroacupuncture (EA) on denervation-induced atrophy in rats. Methods A total of 18 male Sprague-Dawley rats were divided into sham group (n=6), model group (n=6) and EA group (n=6). The latter two groups were clamped right sciatic nerve to establish atrophy model of skeletal muscle. On the second day after modeling, EA group accepted electroacupuncture on right Zusanli (ST36) and Huantiao (GB30) for two weeks. Their gastrocnemius muscles were obtained after intervention, and the wet weight ratio of the gastrocnemius muscles was calculated. The cross-sectional area (CSA) and diameter of muscle fibers were measured after HE staining. The protein expression of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), 70-KD ribosomal protein S6 kinase (p70S6K) and phosphorylated p70S6k (p-p70S6k) was tested with Western blotting. The gene expression of mTOR and p70S6K was detected with real-time quantitative polymerase chain reaction. Results Compared with the sham group, the wet weight ratio of the gastrocnemius muscle, CSA and diameter of the muscle fibers decreased in the model group and EA group (P<0.001), which were more in EA group than in the model group (P<0.01); the protein expression of mTOR, p-mTOR, p70S6K and p-p70S6K increased in the model group (P<0.01), and increased more in EA group (P<0.05); the gene expression of mTOR and p70S6K increased in the model group (P<0.05) , and increased more in EA group (P<0.05).Conclusion Electroacupuncture delays the atrophy of denervated skeletal muscles, which may relate to activation of mTOR/p70S6K signal pathway to impact synthesis of skeletal muscle proteins.

Myostatin promotes distinct responses on protein metabolism of skeletal and cardiac muscle fibers of rodents

Myostatin is a novel negative regulator of skeletal muscle mass. Myostatin expression is also found in heart in a much less extent, but it can be upregulated in pathological conditions, such as heart failure. Myostatin may be involved in inhibiting protein synthesis and/or increasing protein degradation in skeletal and cardiac muscles. Herein, we used cell cultures and isolated muscles from rats to determine protein degradation and synthesis. Muscles incubated with myostatin exhibited an increase in proteolysis with an increase of Atrogin-1, MuRF1 and LC3 genes. Extensor digitorum longus muscles and C2C12 myotubes exhibited a reduction in protein turnover. Cardiomyocytes showed an increase in proteolysis by activating autophagy and the ubiquitin proteasome system, and a decrease in protein synthesis by decreasing P70S6K. The effect of myostatin on protein metabolism is related to fiber type composition, which may be associated to the extent of atrophy mediated effect of myostatin on muscle.

Effect of nutritional intervention on muscle protein synthesis in elderly people: a systematic review / 中华健康管理学杂志

Sarcopenia is a syndrome characterized by progressive and generalised loss of skeletal muscle mass and strength with a risk of adverse outcomes such as physical disability, poor quality of life and increasing risk of infection and mortality. Inadequate intake of nutrients (especially protein) might block muscle protein synthesis, and accelerate age-related sarcopenia progress. Age-related decline in skeletal muscle mass as well as muscle functions has been regarded as major health issues in elderly people. The domestic and foreign literature related to impact of nutritional intervention on muscle protein synthesis and muscle functions in elderly people were retrieved to evaluate the effect of nutritional intervention on muscle protein synthesis and muscle functions in elderly people.

Synthesis of soluble proinsulin in a cell-free protein synthesis system / 生物工程学报

Proinsulin (Pins) is the precursor of insulin. The expression of proinsulin in Escherichia coli forms inclusion body, so that the recombinant protein should be processed with multiple steps to form active insulin. With the development in biotechnology, cell-free protein synthesis (CFPS) system is becoming a valuable tool in protein expression by decoupling the cell growth with protein production, which allows it to express proteins that would interfere with cell physiology. In this study, we synthesized soluble proinsulin in CFPS system in order to establish a new approach for both insulin expression and delivery. The soluble proinsulin was successfully expressed in CFPS system by fusing proinsulin with two types of fluorescent protein. The expression of Pins-mCherry was confirmed by Western blotting analysis, and the Pins-eGFP titer was (12.28±3.45) μg/mL in CFPS system. These results implicated that the proinsulin was expressed partially in soluble form. Here, for the first time, we successfully expressed soluble proinsulin in CFPS system by fluorescent protein fusion. These results provide useful information in developing new insulin expression and delivery method.

Differential Expression Patterns of Leukaemia Associated Genes in Leukaemia Cell Lines Compared to Healthy Controls

Introduction: The phenotype and genotype of cancer cells portray hallmarks of cancer which may have clinical value. Cancer cell lines are ideal models to study and confirm these characteristics. We previously established two subtracted cDNA libraries with differentially expressed genes from an acute myeloid leukaemia patient with poor prognosis (PP) and good prognosis (GP). Objective: To compare gene expression of the leukaemia associated genes with selected biological characteristics in leukaemia cell lines and normal controls. Methodology: Expression of 28 PP genes associated with early fetal/embryonic development, HOX-related genes, hematopoiesis and aerobic glycolysis/ hypoxia genes and 36 GP genes involved in oxidative phosphorylation, protein synthesis, chromatin remodelling and cell motility were examined in B-lymphoid (BV173, Reh and RS4;11) and myeloid (HL-60, K562) leukaemia cell lines after 72h in culture as well as peripheral blood mononuclear cells from healthy controls (N=5) using semi-quantitative polymerase chain reaction (PCR) method. Cell cycle profiles were analysed on flow cytometry while MTT cytotoxicity assay was used to determine drug resistance to epirubicin. Results: Genes expressed significantly higher in B-lymphoid leukaemia cell lines compared to healthy controls were mostly of the GP library i.e. oxidative phosphorylation (3/10), protein synthesis (4/11), chromatin remodelling (3/3) and actin cytoskeleton genes (1/5). Only two genes with significant difference were from the PP library. Cancer associated genes, HSPA9 and PSPH (GP library) and BCAP31 (PP library) were significantly higher in the B-lymphoid leukemia cell lines. No significant difference was observed between myeloid cell lines and healthy controls. This may also be due heterogeneity of cell lines studied. PBMC from healthy controls were not in cell cycle. G2/M profiles and growth curves showed B-lymphoid cells just reaching plateau after 72 hour culture while myeloid cells were declining. IC50 values from cytotoxicity assay revealed myeloid cell lines had an average 13-fold higher drug resistance to epirubicin compared to B-lymphoid cell lines. Only CCL1, was expressed at least two-fold higher in myeloid compared to B-lymphoid cell lines. In contrast, MTRNR2, EEF1A1, PTMA, HLA-DR, C6orf115, PBX3, ENPP4, SELL, and IL3Ra were expressed more than 2-fold higher in B-lymphoid compared to myeloid cell lines studied here. Conclusion: Thus, B-lymphoid leukaemia cell lines here exhibited active, proliferating characteristics closer to GP genes. Higher expression of several genes in B-lymphoid compared to myeloid leukaemia cell lines may be useful markers to study biological differences including drug resistance between lineages.

Hericium erinaceus (Bull.: Fr.) Pers., a medicinal mushroom, activates peripheral nerve regeneration / 中国结合医学杂志

<p><b>OBJECTIVE</b>To study the ability of aqueous extract of Hericium erinaceus mushroom in the treatment of nerve injury following peroneal nerve crush in Sprague-Dawley rats.</p><p><b>METHODS</b>Aqueous extract of Hericium erinaceus was given by daily oral administration following peroneal nerve crush injury in Sprague-Dawley rats. The expression of protein kinase B (Akt) and mitogen-activated protein kinase (MAPK) signaling pathways; and c-Jun and c-Fos genes were studied in dorsal root ganglia (DRG) whereas the activity of protein synthesis was assessed in peroneal nerves by immunohistochemical method.</p><p><b>RESULTS</b>Peripheral nerve injury leads to changes at the axonal site of injury and remotely located DRG containing cell bodies of sensory afferent neurons. Immunofluorescence studies showed that DRG neurons ipsilateral to the crush injury in rats of treated groups expressed higher immunoreactivities for Akt, MAPK, c-Jun and c-Fos as compared with negative control group (P <0.05). The intensity of nuclear ribonucleoprotein in the distal segments of crushed nerves of treated groups was significantly higher than in the negative control group (P <0.05).</p><p><b>CONCLUSION</b>H. erinaceus is capable of promoting peripheral nerve regeneration after injury. Potential signaling pathways include Akt, MAPK, c-Jun, and c-Fos, and protein synthesis have been shown to be involved in its action.</p>

Urinary creatinine as a nutritional and urinary volume marker in sheep fed with tropical or temperate forages / Uso da creatinina urinária como marcador nutricional e de volume urinário em ovinos alimentados com forragem tropical ou temperada

To test the accuracy of creatinine as a marker for estimating urinary volume and its use as a nutritional index, the possible interference of forage intake and forage quality over creatinine excretion was evaluated. For this, sheep were fed different levels of pearl millet (Pennisetum americanum(L) Leeke) or Italian ryegrass (Lolium multiflorum Lam). The experiment consisted of a compilation of digestibility trials (n=6) with pearl millet or Italian ryegrass in completely randomized designs with four replications and four forage levels: 1.5, 2.0, 2.5% (kg dry matter (DM)/ 100 kg of live weight (LW)). The trials were repeated at different periods to evaluate how stable the average metabolic excretion of creatinine is. In each trial, total urine collection was performed individually during a period of 24 hours for five consecutive days and subsequently analyzed by colorimetry for creatinine and purine derivatives. The creatinine excretion was not affected (P>0.05) by forage offer or forage type, but there were period effects (P=0.0001). The average creatinine excretion for both forages was 0.21mmol/kg PV0,75. Linear regressions between the purine derivatives:creatinine index with total excretion of purine derivatives were detected for pearl millet (P<0.0001, R2= 0.64) and Italian ryegrass (P=0.02, R2=0.20). These results demonstrate that creatinine excretion is independent of the type and availability of forage and can be a marker for urinary volume prediction and nutritional measures under grazing systems.

Para testar a precisão da creatinina como marcador para estimativas de volume urinário e índice nutricional, foram avaliadas a possível influência do consumo e a qualidade da forragem sobre esse marcador. Para isso, ovinos foram alimentados com diferentes níveis de milheto (Pennisetum americanum (L) Leeke) ou azevém (Lolium multiflorum Lam). O experimento consistiu de uma compilação de ensaios de digestibilidade (n=6) com milheto ou azevém, em um desenho experimental de blocos completamente ao acaso, com quatro repetições e quatro níveis de forragem: 1,5; 2,0; 2,5% (kg de matéria seca (MS)/ 100kg de peso vivo (PV)). Os ensaios foram repetidos em diferentes períodos, com ambas as forragens, se para avaliar a estabilidade da excreção média de creatinina metabólica. Em cada ensaio, foi coletado o volume total de urina individualmente, durante períodos de 24 horas, por cinco dias consecutivos. Posteriormente, esses ensaios foram analisados por colorimetria para creatinina e derivados de purina. A excreção de creatinina não foi afetada (P>0,05) pelo consumo de forragem ou pelo tipo de forragem, mas foi influenciada pelo período (P=0,0001). A excreção média de creatinina para ambas as forragens foi 0,21mmol/kg PV0,75. Regressões lineares entre os índices derivados de purina:creatinina com a excreção total de derivados de purina foram detectadas para milheto (P<0,0001; R2=0,64) e azevém (P=0,02; R2=0,20). Os resultados demonstraram que a excreção de creatinina é independente do tipo e do consumo de forragem e pode ser usada como marcador preditivo do volume urinário e do status nutricional em sistemas de pastejo.

Nitrogen balance and milk composition of dairy cows fed with urea and soybean meal and two protein levels using sugar cane based diets / Balanço de nitrogênio e composição do leite de vacas alimentadas com ureia e farelo de soja e dois teores de proteína em dietas com cana-de-açúcar

It was evaluated the effect of feeding two levels of crude protein (CP) (low: 142 g CP/kg DM; and high: 156 g CP/kg DM) and two nitrogen sources (soybean meal and urea) to dairy cows using sugar cane as forage on microbial protein synthesis, the composition of the milk nitrogen fraction, nitrogen (N) balance and blood parameters. Twelve Holstein cows with an average milk yield of 22.0 ± 2.3 kg/day, and with 235 ± 40 days in milk were included in this study. The animals were grouped into three balanced and contemporary 4x4 Latin squares for an experimental period of 21 days. On the 15th day of each period, milk and urine samples were collected for microbial protein synthesis determination. Total excretion of urine (L/day), milk urea nitrogen (MUN) and blood urea were higher for the diets with high CP, regardless of the nitrogen source. Nitrogen efficiency was higher for cows fed diets with low CP. Cows in the final third of lactation can be fed diets with reduced CP levels, regardless of the nitrogen source, soybean meal or urea, without influencing the synthesis of microbial protein or the composition of the nitrogen fraction of milk.

Foi avaliado o efeito de dois teores de proteína bruta (PB) (baixa: 142 g de PB/kg de MS e alta: 156 g de PB/kg de MS) e de duas fontes nitrogenadas (farelo de soja e ureia) na dieta de vacas leiteiras, alimentadas com cana-de-açúcar como volumoso sobre a síntese de proteína microbiana, composição da fração nitrogenada do leite, balanço de nitrogênio (N) e parâmetros sanguíneos. Foram utilizadas 12 vacas Holandesas com produção média de 22,0 ± 2,3 kg leite/dia, no terço final de lactação (235 ± 40 dias em lactação), agrupadas em três quadrados latinos balanceados e contemporâneos 4 x 4, com período experimental de 21 dias, dos quais 14 para adaptação às dietas e os demais para coleta de amostras. A excreção total de urina (L/dia), o NUL e a ureia sanguínea foram maiores para as dietas com alta PB, independentemen- te da fonte nitrogenada. A eficiência de utilização do N foi maior para as vacas alimentadas com dietas com baixa PB. Vacas no terço final da lactação podem ser alimentadas com dietas com teores reduzidos de PB, independentemente de a fonte nitrogenada ser farelo de soja ou ureia, sem influenciar a síntese de proteína microbiana, a composição da fração nitrogenada do leite e obter maior eficiência de utilização do N da dieta.

Suplementação com farelo de girassol para ovinos alimentados com silagem de bagaço de sorgo sacarino / Sunflower meal supplementation to wethers fed sorghum bagasse silage

Seis ovinos Texel × Corriedale (43,6±4,4kg de peso corporal (PC)), alimentados ad libitum com silagem de bagaço de sorgo sacarino (Sorghum bicolor (L.) Moench ssp. saccharatum), foram usados em um experimento em duplo Quadrado Latino 3×3 para avaliar o efeito nutricional da suplementação com níveis de farelo de girassol (0, 7 ou 14g kg-1 de PC). Uma solução (8%, p/v) de ureia e sulfato de amônio (9:1, respectivamente) foi misturada à silagem no momento do fornecimento aos animais, em todos os tratamentos, numa proporção de 50ml de solução kg-1 de silagem. O consumo total de matéria seca (MS), matéria orgânica (MO), fibra em detergente neutro (FDN) e de carboidratos não fibrosos (CNF), assim como o consumo de MS da silagem e de MO digestível, foram positivamente afetados (P<0,05) pelo aumento da suplementação com farelo de girassol. A digestibilidade da FDN não foi afetada pelos tratamentos, enquanto que a digestibilidade aparente da MS e da MO foi linearmente (P<0,05) incrementada pela suplementação. A eficiência de síntese proteica microbiana ruminal não foi influenciada pelos tratamentos, enquanto que o consumo, a digestibilidade, a excreção urinária e a retenção de N, assim como a síntese de proteína microbiana ruminal, foram linearmente e positivamente afetados pela suplementação (P<0,05). Em conclusão, a suplementação com farelo de girassol impacta positivamente a utilização da silagem de bagaço de sorgo sacarino e a oferta de nutrientes para ovinos.

Six Texel × Polwarth (43.6±4.4kg of body weight (BW)) wethers fed ad libitum sorghum bagasse silage (Sorghum bicolor (L.) Moench ssp. saccharatum) were used in a replicated 3×3 Latin Square experiment to evaluate the nutritional effect of supplementing levels of sunflower meal (0, 7 or 14g kg-1 de BW). In all treatments the urea plus ammonium sulphate (9:1, respectively) solution (8%, p/v) was mixed to silage at feeding at a rate of 50ml k-1g of silage. Total dry matter (DM), organic matter (OM) neutral detergent fibre (NDF) and non-fibre carbohydrates (NFC) intake, as well as DM intake from silage and digestible OM intake were positively affected (P<0.05) by increased levels of sunflower meal supplementation. The NDF digestibility was not affected by treatments whereas the apparent DM and OM digestibility were linearly increased (P<0.05) due supplementation. The efficiency of ruminal microbial protein synthesis was not affected by treatments whereas N intake, digestibility, urinary excretion and retention as well as rumen microbial protein synthesis increased linearly (P<0.05) at increased levels of supplementation. In conclusion, sunflower meal supplementation positively impacts sorghum silage utilization and nutrients supply to wethers.

Efeitos da suplementação crônica de L-arginina sobre a expressão de proteínas envolvidas na regulação da síntese proteica muscular em ratos treinados em exercícios de alta intensidade / Effects of chronic supplementation of L-arginine on the expression of proteins involved in the regulation of muscle protein synthesis in muscle of trained rats in high-intense Exercise

A arginina é um aminoácido condicionalmente essencial que participa de inúmeras reações metabólicas no organismo como, por exemplo, o ciclo da uréia, a síntese de creatina e a geração de óxido nítrico (NO). Além dessas funções a arginina é associada, com a sensibilidade à insulina, a secreção de GH e mais recentemente com a síntese protéica muscular. O objetivo deste trabalho foi investigar o efeito da suplementação via oral crônica de L-arginina sobre a síntese protéica muscular, pela via da mTOR, a fim de contribuir com as novas discussões científicas acerca deste aminoácido de ampla atuação. Métodos: Foram utilizados ratos wistar machos adultos com cerca de 200g de peso corporal divididos em quatro grupos de quatorze animais denominados na seguinte forma: Arginina Treinado (AT), Arginina Sedentário (AS), Dieta-Controle Treinado (CT) e Dieta-Controle Sedentário (CS). Ambas as dietas foram elaboradas com base das recomendações da AIN-93, sendo que a dieta enriquecida com arginina recebeu acréscimo de 2% deste aminoácido e a dieta controle recebeu um mix de aminoácidos não essenciais para garantir que ambas fossem isonitrogenadas e isocalóricas e as proporções de aminoácidos presente nas rações foi conferida por aminograma. O treinamento dos animais consistiu em exercício anaeróbio com sessões que eram compostas de 4 séries de 10 saltos com um minuto de descanso entre estas em tanque de água. Os saltos eram desempenhados com carga de 50% do peso corporal acoplado ao tórax dos animais na freqüência de 5 dias por semana por 6 semanas. A evolução da massa corporal dos animais bem como o consumo de ração foram avaliadas três vezes por semana e estimada uma média semanal. Foram realizados testes de tolerância oral à glicose (OGTT) e tolerância à insulina (ITT) no início e ao final do experimento em todos os animais para avaliar alterações na sensibilidade à insulina. Após 72hs da última sessão de treinamento os animais foram anestesiados para infusão de insulina, coleta dos músculos gastrocnêmio e plantar, fígado, sangue e eutanasiados conforme protocolo aprovado pelo CEA-USP. As análises bioquímicas foram determinações séricas de insulina, GH, IGF-1 e a proteína transportadora de IGF-1 (IGFBP-3), glicose plasmática, uréia e creatinina séricas, IGF-1 muscular e hepático por kits comerciais de tecnologia multiplex Luminex e aminograma sérico por cromatografia. As análises moleculares foram realizadas para as proteínas chaves envolvidas na via de síntese protéica muscular em sua forma total e fosforilada, sendo estas: IRS-1, Akt, mTOR, 4E-BP1 e p70S6K determinadas por método de western blotting. Resultados: Não foram encontradas diferenças estatisticamente significativas nos parâmetros avaliados com exceção da creatinina que se mostrou mais elevada nos grupos suplementados com arginina. A suplementação de arginina, nas concentrações administradas, bem como o exercício de alta intensidade pelo período determinado não foram capazes de alterar a expressão das proteínas envolvidas na regulação de síntese protéica muscular de ratos nem a sensibilidade celular à insulina. Conclusão: não houve aumento da síntese protéica muscular com a suplementação de arginina, nestas condições experimentais

The arginine is an amino acid conditionally essential that participates in innumerous metabolic reactions in the body like, for instance, the urea cycle, the synthesis of creatine and production of nitric oxide (NO). Besides those functions the arginine is associated, with the insulin sensitivity, GH secretion and most recently with muscle protein synthesis. The aim of this work was to investigate the effect of L-arginine chronic oral supplementation on the muscle protein synthesis, through mTOR pathway, in order to contribute with new scientific discussions about this broad action amino acid. Methods: Wistar male adult rats were used with about 200g body weight distribute into four groups of fourteen animals named this way: Trained Arginine (TA), sedentary Arginine (SA), Trained Diet-Control (TC) and Sedentary Diet-control (SC). Both diets were elaborated based on the AIN-93 recommendations, considering that the enriched diet with arginine was added 2% of this amino acid and the control diet received a mix of non-essential amino acid in order to ensure that both were isonitrogenous and isocaloric and the proportions of present amino acids in the rations have been checked through aminogram. The animals training consisted of anaerobic exercise with sections composed by four jump series, with one minute rest among these in a PVC cube water. The jumps were performed with a load of 50% of their body weight attached in the animal's trunk, five days a week over six weeks. The animals' body weight evolution as well as the food intake were evaluated three times a week in order to figure a weekly average. Oral glucose test tolerance (OGTT) and insulin test tolerance (ITT) have been done in the beginning and in the end of the experiment in all animals to evaluate insulin sensitive changes. The animals were anesthetized to insulin infusion, gastrocnemic and plantaris muscles, liver and blood collects 72 hrs after the last training section and afterwards sacrificed according to CEA-USP approved protocol. The biochemical analysis were blood determination of insulin, GH, IGF-1 and its binding protein 3 (IGFBP-3), glucose, urea and creatinine, and muscle and liver IGF-1 through commercial kits of multiplex Luminex technology and seric aminogram through chromatography. The molecular analysis were performed for the key proteins of the muscle protein synthesis pathway in its total and phosphorylated form: IRS-1, Akt-1, mTOR, 4E-BP1 and p70S6K determined by western blotting method. Results: Significant statistical differences were not found to all evaluated biomarkers in this experiment except for creatinine which was more elevated in groups supplemented with arginine. The arginine supplementation, in these given doses, as well as the high-intense exercise, failed in stimulate both the expression of the proteins involved in the muscle protein synthesis regulation and the insulin sensitivity in the rats in this condition. Conclusion: There hasn't been any increase in the muscle protein synthesis with arginine supplementation, in these experimental conditions

Research progress and prospect of human β-defensin 3 and its derivatives in ophthalmology / 中华实验眼科杂志

Currently,antimicrobial resistance has become an urgent global public health problem.Human defensin,a family of innate immune peptides,plays an important role in the control of infectious disease because of its low resistance to microbiological infection.However,so far there is seldom report on industrial production of bioactive defensin.Human β-defensin 3 (HBD3) is a member of human innate antimicrobial peptides.It has broad-spectrum antimicrobial activity and function in a variety of inflammation diseases.In addition,HBD3 is considered as a good candidate for antibiotics,because it has a strong killing activity to antibiotics-resistant microbes and resistance to highsalt environment,it is especially valuable for the treatment of ocular surface inflammatory diseases.This review covers the in vitro research on the production of HBD3,optimization of structure-activity and its antimicrobial potential for ocular surface infection,which may provide a supportive evidence for the commercial production of HBD3 and other antimicrobial peptides in biopharmaceutical area.

Avaliação nutricional da polpa de maçã como suplementação energética para bovinos / Nutritional evaluation of apple pulp as energetically supplementation to cattle

Objetivou-se avaliar o efeito da suplementação com polpa de maçã, em substituição ao grão de milho, para bovinos ingerindo azevém anual verde. Os tratamentos experimentais foram pasto de azevém à vontade (AZ) ou suplementado com 0,5% do peso vivo de MS constituída: de grão de milho (AM), de uma mistura de grão de milho + polpa de maçã (50:50; AMP) ou de polpa de maçã (AP). Os animais utilizados foram quatro novilhos machos, castrados, com peso vivo médio de 219±22,1kg, dotados de cânula ruminal e distribuídos em um quadrado latino 4×4, com períodos de 16 dias (10 de adaptação e 6 de coleta). O consumo voluntário de MO da forragem (71,5g kg-1 PV0,75) não se alterou com o uso de suplementação nem com o tipo de suplemento, mas o consumo total de MO e de energia metabolizável aumentaram, respectivamente, 17,4g kg-1 PV0,75 e 9,8MJ dia-1 nos animais suplementados, sem que houvesse diferença entre os animais recebendo os diferentes tipos de suplementação. O pH ruminal (6,7), o valor energético das dietas (média = 12,1MJ kg-1 MS) e a síntese de proteína microbiana (média = 414g dia-1) não se alteraram com o fornecimento da suplementação energética nem com o tipo desta. Entretanto, a digestibilidade do FDA (-10%) e o consumo de MO digestível (-9,7g kg-1 PV0,75) foram inferiores nos animais ingerindo somente polpa, em comparação aos demais tipos de suplementação. A substituição de até 50% da MS do grão de milho por polpa de maçã, para bovinos ingerindo pastos de azevém, é uma prática segura quando se objetiva aumentar a ingestão de MO digestível.

The aim of this work was to evaluate the effect of supplementation with apple pulp to heifers receiving fresh Italian ryegrass. The treatments were fresh Italian ryegrass (R) ad libidum or supplemented with 0.5% of DM of body weight as: corn ground (RC), corn ground + apple pulp (50:50; RCP) or apple pulp (RP). Four Holstein steers (mean LW of 219±22.1kg) with permanent ruminal cannulae, housed in metabolic cages were assigned in a 4×4 latin square experiment with periods of 16 days (10 for adaptation and 6 for measurements). The OM voluntary intake of herbage was similar in heifers with or without supplementation (average = 71.5g kg-1 BW0,75), but total OM intake and metabolisable energy intake increased, respectively, 17.4g kg-1 PV0.75 e 9.8MJ day-1 in supplemented heifers compared with the no supplemented ones. The ruminal pH (average = 6.7), the energetically values of diets (average = 12.0MJ kg-1 DM) and microbial protein synthesis (average 414g day-1) did not change with supplementation. However the ADF digestibility (-10%) and digestible OM intake (-9.7g kg-1 BW0.75) were lower in animals receiving apple pulp only comparing with other supplement types. In conclusion, to improve the digestible OM intake apple pulp can be used to replace until 50% of DM of corn ground to heifers eating fresh Italian ryegrass.

Digestibility and microbial efficiency in steers fed diets based on corn silage hybrids and concentrate levels / Digestibilidade e eficiência microbiana em novilhos alimentados com dietas à base de silagem de híbridos de milho e níveis de concentrado

A trial involving a 2x2 factorial design was conducted to evaluate the effect of corn silage hybrids and concentrate levels (25 and 50%) on intake and digestibility of nutrients, ruminal characteristics and microbial efficiency in steers. Four ruminal and abomasal cannulated steers (512±25kg of birth weight), were used in a 4×4 Latin square design. Treatments consisted of 75% silage A + 25% concentrate; 50% silage A + 50% concentrate; 75% silage B + 25% concentrate; and 50% silage B + 50% concentrate on dry matter (DM) basis. There were no differences in the intakes of DM, organic matter (OM), crude protein, and ether extract. The intake of non fiber carbohydrates and total digestible nutrients were positively affected by concentrate levels. The digestibility of DM and OM were also positively affected by concentrate levels. There were no effects of treatments on ruminal pH values, ruminal ammonia-N, and microbial efficiency.

Avaliou-se, em um esquema fatorial 2x2, o efeito de silagem de milho de diferentes híbridos e da porcentagem de concentrado (25 ou 50%) sobre o consumo e a digestibilidade de nutrientes, os parâmetros ruminais e a síntese de proteína microbiana em bovinos. Utilizaram-se quatro novilhos cruzados (HxZ) - (512±25kg) -, fistulados no rúmen e no abomaso, os quais foram distribuídos em um quadrado latino 4x4. Os tratamentos consistiram em 75% de silagem A + 25% de concentrado; 50% de silagem A + 50% de concentrado; 75% de silagem B + 25% de concentrado; e 50% de silagem B + 50% de concentrado em % da matéria seca (MS). Não houve diferença entre os consumos de MS, matéria orgânica (MO), proteína bruta e extrato etéreo, e houve efeito positivo de concentrado sobre os carboidratos não fibrosos e nutrientes digestíveis totais. A digestibilidade da MS e da MO foi positivamente influenciada pela porcentagem de concentrado. Não houve efeito de tratamento sobre o pH e a concentração de amônia ruminais, bem como sobre a eficiência de síntese microbiana.

Effect of Intensity of Unconditional Stimulus on Reconsolidation of Contextual Fear Memory

Memory reconsolidation is ubiquitous across species and various memory tasks. It is a dynamic process in which memory is modified and/or updated. In experimental conditions, memory reconsolidation is usually characterized by the fact that the consolidated memory is disrupted by a combination of memory reactivation and inhibition of protein synthesis. However, under some experimental conditions, the reactivated memory is not disrupted by inhibition of protein synthesis. This so called "boundary condition" of reconsolidation may be related to memory strength. In Pavlovian fear conditioning, the intensity of unconditional stimulus (US) determines the strength of the fear memory. In this study, we examined the effect of the intensity of US on the reconsolidation of contextual fear memory. Strong contextual fear memory, which is conditioned with strong US, is not disrupted by inhibition of protein synthesis after its reactivation; however, a weak fear memory is often disrupted. This suggests that a US of strong intensity can inhibit reconsolidation of contextual fear memory.