Molecular characterization of ANKRD1 in rhabdomyosarcoma cell lines: expression, localization, and proteasomal degradation.

Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy in children and adolescents. Respecting the age of the patients and the tumor aggressiveness, investigation of the molecular mechanisms of RMS tumorigenesis is directed toward the identification of novel therapeutic targets. To contribute to a better understanding of the molecular pathology of RMS, we investigated ankyrin repeat domain 1 (ANKRD1), designated as a potential marker for differential diagnostics. In this study, we used three RMS cell lines (SJRH30, RD, and HS-729) to assess its expression profile, intracellular localization, and turnover. They express wild-type ANKRD1, as judged by the sequencing of the open reading frame. Each cell line expressed a different amount of ANKRD1 protein, although the transcript level was similar. According to western blot analysis, ANKRD1 protein was expressed at detectable levels in the SJRH30 and RD cells (SJRH30 > RD), but not in the HS-729, even after immunoprecipitation. Immunocytochemistry revealed nuclear and cytoplasmic localization of ANKRD1 in all examined cell lines. Moreover, the punctate pattern of ANKRD1 staining in the nuclei of RD and HS-729 cells overlapped with coilin, indicating its association with Cajal bodies. We have shown that RMS cells are not able to overexpress ANKRD1 protein, which can be attributed to its proteasomal degradation. The unsuccessful attempt to overexpress ANKRD1 in RMS cells indicates the possibility that its overexpression may have detrimental effects for RMS cells and opens a window for further research into its role in RMS pathogenesis and for potential therapeutic targeting.

Proteomics in bovine semitendinosus muscle to assess emerging strategies based on papain injection and ultrasounds on meat tenderization process.

The impact of papain and/or ultrasound treatments on tenderization of semitendinosus muscle through a proteomic approach was studied. Sixteen bovine muscles were submitted to the following treatments: aging at 3 °C (Control), papain injection (PI), ultrasound (US), PI followed by US (PIUS) and US followed by PI (USPI). pH, myofibrillar fragmentation indices (MFI), soluble collagen, texture profile and changes of myofibrillar proteins were investigated after 2, 24, 48 and 96 h of storage. The highest MFI and soluble collagen content were found in PI, PIUS and USPI samples while control samples showed the lowest values. PI samples showed the lowest WBSF and hardness values until 48 h of storage while at 96 h meat from USPI treatment showed WBSF value comparable to PI treatment. The lowest values of cohesiveness, gumminess and chewiness were found in PI samples during all storage times. Proteomic analysis revealed a different quantity and expression of proteins among tenderization treatments. US treatment did not exhibit a significant ability to degrade muscle proteins, while, all treatments containing papain, showed a greater ability to hydrolyse and degrade myofibrillar proteins. PI promoted intense proteolysis leading to an early tenderization process; on the contrary, in PIUS and USPI treatments the sequence of treatments was relevant on meat tenderization. Particularly, USPI treatment, after 96 h, reached the same improvement in tenderness of enzymatic treatment but with slower hydrolysing rate; this could be determinant to preserve textural structure.

Identification of Subproteomic Markers for Skeletal Muscle Profiling.

The biochemical and cell biological profiling of contractile fiber types and subcellular structures plays a central role in basic and applied myology. Mass spectrometry-based proteomics presents an ideal approach for the systematic identification of proteomic and subproteomic markers. These representative components of fast versus slow muscle fibers and their subcellular fractions are highly useful for in-depth surveys of skeletal muscle adaptations to physiological challenges, as well as the improvement of diagnostic, prognostic, and therapy-monitoring methodologies in muscle pathology. This chapter outlines the identification of subproteomic markers for skeletal muscle profiling based on bottom-up and top-down approaches, including fluorescence two-dimensional difference gel electrophoresis (2D-DIGE).

Effects of high-intensity gymnastics on skeletal muscle proteins / Efeitos da ginástica de alta intensidade sobre as proteínas musculares esqueléticas / Efectos de la gimnasia de alta intensidad en las proteínas musculares esqueléticas

ABSTRACT Introduction: Although the current method of muscle stretching in gymnastics teaching in colleges and universities can reduce sports fatigue, it has been shown to have little effect on the well-being of athletes because it requires a long recovery time from psychological fatigue. Progressive muscle relaxation training is a method that uses the basic principle of sympathetic nerve activity to reduce the impact of negative emotions psychologically and relieve fatigue physiologically, requiring a further study of its impact on muscle protein. Objective: Explore the effect of high-intensity gymnastics on skeletal muscle protein and study the progressive muscle relaxation training method post-workout adjustment. Methods: After three weeks of training, excluding the standard deviations in the experimental data caused by the athletes' irregular movements, the athletes' blood lactate content and heart rate were counted and recorded. The collected data were analyzed using Excel software to integrate and compare the data using the T-test method. Results: After exercise training, the skeletal muscle function indices of the subjects increased to different degrees. From the point of view of heart rate recovery efficiency, the rate of heart rate decline of progressive relaxation training was higher than that of the two groups, and the degree of fluctuation was lower than that of the two groups, indicating that the level of recovery in heart rate of progressive relaxation training was better. Conclusion: The action of the high-intensity gymnastics team has a good effect on improving the athletes' skeletal muscle and skeletal muscle proteins. Post-exercise conditioning training plays an important role in athletes' physical recovery. Level of evidence II; Therapeutic studies - investigation of treatment outcomes.

RESUMO Introdução: Embora o método de alongamento muscular atual no ensino de ginástica em faculdades e universidades consiga reduzir a fadiga esportiva, tem se mostrado pouco eficaz no bem-estar dos atletas por exigir grande tempo de recuperação da fadiga psicológica. O treinamento progressivo de relaxamento muscular é um método que usa o princípio básico da atividade nervosa simpática para reduzir o impacto das emoções negativas psicologicamente e aliviar a fadiga fisiologicamente, necessitando de mais estudos do seu impacto sobre a proteína muscular. Objetivo: Explorar o efeito da ginástica de alta intensidade sobre as proteínas musculares esqueléticas e estudar o método de treinamento progressivo de relaxamento muscular no ajuste pós-treino. Métodos: Após 3 semanas de treinamento, excluídos os desvios-padrão nos dados experimentais causados pelos movimentos irregulares dos atletas, foram contabilizados e registrados os conteúdos de lactato sanguíneo e frequência cardíaca dos atletas. Analisou-se os dados coletados, com o software Excel, para integrar e comparar os dados pelo método de teste-T. Resultados: Após o treinamento do exercício, os índices de função muscular esquelética dos sujeitos aumentaram em diferentes graus. Do ponto de vista da eficiência da recuperação da frequência cardíaca, a taxa de declínio da frequência cardíaca do treinamento de relaxamento progressivo foi maior do que a dos dois grupos, o grau de flutuação foi menor do que o dos dois grupos, indicando que o nível de recuperação na frequência cardíaca do treinamento de relaxamento progressivo foi melhor. Conclusão: A ação da equipe de ginástica de alta intensidade tem um bom efeito na melhoria do músculo esquelético e das proteínas musculares esqueléticas dos atletas. O treinamento de condicionamento pós-exercício desempenha um papel importante na recuperação física dos atletas. Nível de evidência II; Estudos terapêuticos - investigação dos resultados do tratamento.

RESUMEN Introducción: Aunque el método actual de elongación muscular en la enseñanza de la gimnasia en colegios y universidades consigue reducir la fatiga deportiva, se ha demostrado que tiene poco efecto en el bienestar de los atletas porque requiere un largo tiempo de recuperación de la fatiga psicológica. El entrenamiento de la relajación muscular progresiva es un método que utiliza el principio básico de la actividad nerviosa simpática para reducir el impacto de las emociones negativas desde el punto de vista psicológico y aliviar la fatiga desde el punto de vista fisiológico, lo que requiere un estudio más profundo de su impacto en la proteína muscular. Objetivo: Explorar el efecto de la gimnasia de alta intensidad sobre la proteína del músculo esquelético y estudiar el método de entrenamiento de relajación muscular progresiva en el ajuste posterior al entrenamiento. Métodos: Después de 3 semanas de entrenamiento, excluyendo las desviaciones estándar en los datos experimentales causadas por los movimientos irregulares de los atletas, se contó y registró el contenido de lactato en sangre y la frecuencia cardíaca de los atletas. Los datos recogidos se analizaron, con el programa informático Excel, para integrar y comparar los datos mediante el método de la prueba T. Resultados: Tras el entrenamiento con ejercicios, los índices de función del músculo esquelético de los sujetos aumentaron en diferentes grados. Desde el punto de vista de la eficacia de la recuperación de la frecuencia cardíaca, el índice de disminución de la frecuencia cardíaca del entrenamiento de relajación progresiva fue mayor que el de los dos grupos, el grado de fluctuación fue menor que el de los dos grupos, lo que indica que el nivel de recuperación de la frecuencia cardíaca del entrenamiento de relajación progresiva fue mejor. Conclusión: La acción del equipo de gimnasia de alta intensidad tiene un buen efecto en la mejora del músculo esquelético y de las proteínas del músculo esquelético de los atletas. El entrenamiento de acondicionamiento posterior al ejercicio desempeña un papel importante en la recuperación física de los deportistas. Nivel de evidencia II; Estudios terapéuticos - investigación de los resultados del tratamiento.

[The role of expression of monocarboxylates of the first and fourth types (MCT1, MCT4) by tumor and stromal cells of prostate cancer in determining the prognosis and the efficiency of definitive treatment].

AIM: A search for new methods for diagnosing clinically significant prostate cancer is of importance due to the insufficient accuracy of modern methods in detecting aggressive tumors. One of the promising opportunities for the early diagnosis of clinically significant prostate cancer is the assessment of the glycolytic profile of the tumor by determining the expression of monocarboxylates (MCT) types 1 and 4 in tumor cells, as well as in adjacent stromal cells. MATERIALS AND METHODS: An analysis of patients of who underwent radical prostatectomy at the Institute of Urology and Reproductive Health of Sechenov University from 2015 to 2017 was carried out. The patients with histologically confirmed prostate adenocarcinoma were included in the study. Among them, the presence or absence of biochemical recurrence during the first year was studied. An immunohistochemical (IHC) study of postoperative specimen was performed to determine the expression of MCT1 and MCT4 by tumor and stromal cells. The correlation between the intensity of their expression and the risk of biochemical recurrence and the tumor characteristics was evaluated. RESULTS: High membrane expression of MCT1 directly correlated with high stromal expression of MCT4 (r=0.314, p<0.003). A significant direct correlation was found between the predominance of stromal expression of MCT4 over membrane expression and biochemical recurrence (r=0.403, p<0.001), as well as a high ISUP group (4 and 5) (r=0.294, p=0.005). CONCLUSIONS: Determination of the level of expression of type 1 and 4 monocarboxylate transporters in adenocarcinoma cells and tumor stromal cells can become an effective tool for risk stratification, and may also predict the biological behaviors of the prostate cancer and the efficiency of definitive treatment.

Coexpression of MCT1 and MCT4 in ALK-positive Anaplastic Large Cell Lymphoma: Diagnostic and Therapeutic Implications.

In solid tumors, glycolytic cancer or stromal cells export lactates through monocarboxylate transporter (MCT) 4, while oxidative cancer or stromal cells take up lactates as metabolic fuels or signaling molecules through MCT1. CD147 acts as a chaperone of MCT1 or MCT4. Unlike solid tumors, malignant lymphomas have a peculiar tumor microenvironment. To investigate the metabolic phenotype of malignant lymphoma associated with lactate transport, we analyzed immunohistochemical expressions of MCT1, MCT4, and CD147 in 247 cases of various malignant lymphomas. Surprisingly, both MCT1 and MCT4 were diffusely expressed on tumor cell membranes in all cases (11/11, 100%) of anaplastic lymphoma kinase (ALK) (+) anaplastic large cell lymphoma (ALCL). In contrast, only MCT1 was diffusely expressed in tumor cells of ALK(-) ALCL, as well as in B-cell, natural killer/T-cell, T-cell, and classic Hodgkin lymphomas. In these lymphomas, MCT4 expression was mostly localized to adjacent stromal cells. The pattern of diffuse membranous MCT1 and partial MCT4 expressions in tumor cells was observed in 1 case each of peripheral T-cell lymphoma (1/15, 6.7%) and multiple myeloma (1/34, 2.9%). CD147 was diffusely expressed in all types of lymphoma tumor and/or stromal cells. In conclusion, ALK(+) ALCL has a unique metabolism showing high coexpression of MCT1 and MCT4 in tumor cells. Because only ALK(+) ALCL overexpresses MCT4, immunostaining for MCT4 together with ALK is very useful for differential diagnosis from ALK(-) ALCL or peripheral T-cell lymphoma. Moreover, dual targeting against MCT1 and MCT4 would be an appropriate therapeutic approach for ALK(+) ALCL.

Proteomics analysis to investigate the impact of diversified thermal processing on meat tenderness in Hengshan goat meat.

During the thermal processing, proteins of Hengshan goat meat undergo structural modifications such as degradation, oxidation and denaturation, ultimately affect the palatability and acceptability. The results of several objective metrics demonstrated that thermal processing exhibited significant impacts on the tenderness of goat meat. The 551, 84, 72, and 121 proteins were identified in the control and thermal processed groups (boiled, steamed, and roasted), respectively. Compared with the control group, the 101, 98, and 109 differentially-expressed proteins were explored in the treatment groups. Furthermore, the functions of metabolic and skeletal muscle proteome were investigated and discussed. Sensory evaluation and proteomics analysis showed that steaming and boiling treatment had no significant effect on the tenderness of goat meat, while roasting significantly reduced the tenderness, indicating that the available thermal processing methods to ensure the tenderness of goat meat were steaming and boiling treatments. Thus, the established proteomics database of goat meat provided the valuable reference for rational selection of thermal processing methods.

High YAP and TEAD4 immunolabelings are associated with poor prognosis in patients with gallbladder cancer.

Yes-associated protein (YAP) and TEA domain-containing sequence-specific transcription factors 4 (TEAD4) are essential components of the Hippo pathway. Abnormal regulation of the Hippo pathway contributes to the progression and metastasis of many cancer types. However, their clinicopathologic and prognostic significances have not been studied in gallbladder cancers. Here, we systematically evaluated the YAP and TEAD4 immunolabelings and their association with clinicopathologic characteristics and survival outcomes using 212 specimens of surgically resected gallbladder cancers. High YAP and TEAD4 immunolabelings were identified in 70 (33%) cases and were associated with infiltrative growth pattern, poor differentiation, perineural invasion, and advanced pT classification and AJCC stage. High YAP immunolabeling was significantly associated with high TEAD4 immunolabeling (p < 0.001). High immunolabeling levels of YAP or TEAD4 alone and the combined YAPhigh TEAD4high group were significantly associated with poor survival in both univariate (p < 0.001) and multivariate analyses (HR = 2.358; 95% CI, 1.369-4.061; p = 0.002). Therefore, the YAP and TEAD4 immunolabelings are associated with aggressive behavior of gallbladder cancers and may be useful as a prognostic indicator in patients with surgically resected gallbladder cancer.

Histological and biochemical evaluation of skeletal muscle in the two salmonid species Coregonus maraena and Oncorhynchus mykiss.

The growth of fishes and their metabolism is highly variable in fish species and is an indicator for fish fitness. Therefore, somatic growth, as a main biological process, is ecologically and economically significant. The growth differences of two closely related salmonids, rainbow trout (Oncorhynchus mykiss) and maraena whitefsh (Coregonus maraena), have not been adequately studied as a comparative study and are therefore insufficiently understood. For this reason, our aim was to examine muscle growth in more detail and provide a first complex insight into the growth and muscle metabolism of these two fish species at slaughter size. In addition to skeletal muscle composition (including nuclear counting and staining of stem and progenitor cells), biochemical characteristics, and enzyme activity (creatine kinase, lactate dehydrogenase, isocitrate dehydrogenase) of rainbow trout and maraena whitefish were determined. Our results indicate that red muscle contains cells with a smaller diameter compared to white muscle and those fibres had more stem and progenitor cells as a proportion of total nuclei. Interestingly, numerous interspecies differences were identified; in rainbow trout muscle RNA content, intermediate fibres and fibre diameter and in whitefish red muscle cross-sectional area, creatine kinase activity were higher compared to the other species at slaughter weight. The proportional reduction in red muscle area, accompanied by an increase in DNA content and a lower activity of creatine kinase, exhibited a higher degree of hypertrophic growth in rainbow trout compared to maraena whitefish, which makes this species particularly successful as an aquaculture species.

The effect of a chemotherapy drug cocktail on myotube morphology, myofibrillar protein abundance, and substrate availability.

Cachexia, a condition prevalent in many chronically ill patients, is characterized by weight loss, fatigue, and decreases in muscle mass and function. Cachexia is associated with tumor burden and disease-related malnutrition, but other studies implicate chemotherapy as being causative. We investigated the effects of a chemotherapy drug cocktail on myofibrillar protein abundance and synthesis, anabolic signaling mechanisms, and substrate availability. On day 4 of differentiation, L6 myotubes were treated with vehicle (1.4 µl/ml DMSO) or a chemotherapy drug cocktail (a mixture of cisplatin [20 µg/ml], leucovorin [10 µg/ml], and 5-fluorouracil [5-FLU; 50 µg/ml]) for 24-72 h. Compared to myotubes treated with vehicle, those treated with the drug cocktail showed 50%-80% reductions in the abundance of myofibrillar proteins, including myosin heavy chain-1, troponin, and tropomyosin (p < 0.05). Cells treated with only a mixture of cisplatin and 5-FLU had identical reductions in myofibrillar protein abundance. Myotubes treated with the drug cocktail also showed >50% reductions in the phosphorylation of AKTSer473 and of mTORC1 substrates ribosomal protein S6Ser235/236 , its kinase S6K1Thr389 and eukaryotic translation initiation factor 4E-binding protein 1 (all p < 0.05). Drug treatment impaired peptide chain initiation in myofibrillar protein fractions and insulin-stimulated glucose uptake (p = 0.06) but increased the expression of autophagy markers beclin-1 and microtubule-associated proteins 1A/1B light chain 3B (p < 0.05), and of apoptotic marker, cleaved caspase 3 (p < 0.05). Drug treatment reduced the expression of mitochondrial markers cytochrome oxidase and succinate dehydrogenase (p < 0.05). The observed profound negative effects of this chemotherapy drug cocktail on myotubes underlie a need for approaches that can reduce the negative effects of these drugs on muscle metabolism.

Assessing the reproducibility of labelled antibody binding in quantitative multiplexed immuno-mass spectrometry imaging.

Immuno-mass spectrometry imaging (iMSI) uses laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) to determine the spatial expression of biomolecules in tissue sections following immunolabelling with antibodies conjugated to a metal reporter. As with all immunolabelling techniques, the binding efficiency of multiplexed staining can be affected by a number of factors including epitope blocking and other forms of steric hindrance. To date, the effects on the binding of metal-conjugated antibodies to their epitopes in a multiplexed analysis have yet to be quantitatively explored by iMSI. Here we describe a protocol to investigate the effects of multiplexing on reproducible binding using the muscle proteins, dystrophin, sarcospan, and myosin as a model, with antibodies conjugated with Maxpar® reagents before histological application to murine quadriceps sections using standard immunolabelling protocols and imaging with LA-ICP-MS. The antibodies were each individually applied to eight sections, and multiplexed to another eight sections. The average concentrations of the lanthanide analytes were determined, before statistical analyses found there was no significant difference between the individual and multiplexed application of the antibodies. These analyses provide a framework for ensuring reproducibility of antibody binding during multiplexed iMSI, which will allow quantitative exploration of protein-protein interactions and provide a greater understanding of fundamental biological processes during healthy and diseased states.

Changes in the digestion properties and protein conformation of sturgeon myofibrillar protein treated by low temperature vacuum heating during in vitro digestion.

The digestion properties of sturgeon myofibrillar protein (MF) treated by low temperature vacuum heating (LTVH) at different processing temperatures (50, 60 and 70 °C) and times (15 and 30 min) were studied and compared with those of sturgeon MF treated by traditional cooking (TC). The results showed that as the temperature and time increased, the protein digestibility decreased, whereas the particle size and protein aggregation increased. It was observed that the band intensity of myosin heavy chain and myosin heavy chain 7 weakened; however, the band intensity of actin showed little change. MALDI-TOF-MS analysis revealed that the digested products of the samples treated by LTVH had a larger proportion of 750-1000 Da peptides than those treated by TC, which was consistent with the trend of the number of unique peptides identified in each group. Fourier transmission infrared (FT-IR) spectroscopy showed that the contents of α-helices and ß-sheets exhibited negative and positive correlations with the temperature, respectively. Overall, compared to TC, LTVH can relieve the heat stress of protein conformation, reduce protein aggregation to improve the accessibility of the protein to digestive protease, and increase digestibility.

Performance of different portable and hand-held near-infrared spectrometers for predicting beef composition and quality characteristics in the abattoir without meat sampling.

The availability of portable and handheld NIR instruments on the market opens up new possibilities in meat analysis. However, there is lack of research comparing different NIR instruments for evaluating beef characteristics from spectra obtained directly on the meat surface. Our aim, therefore, was to build and test calibration and prediction models for predicting beef characteristics, and to compare the performances of three NIR instruments differing in size and characteristics: a transportable visible-NIR spectrometer (Vis-NIRS), a portable (NIRS), and a hand-held Micro-NIRS. Spectra were collected from 178 beef samples (Longissimus thoracis muscle) from the meat surface in the abattoir. The spectra were subjected to different mathematical pretreatments then partial least square regressions. The results showed that all instruments predicted dry matter, protein and lipids with R2VAL 0.23 to 0.70; pH and cooking loss R2VAL 0.19 to 0.25; and color R2VAL 0.35 to 0.77. Overall, the prediction performances of the three instruments were similar, although Micro-NIRS performed better in some respects.

TRIM63 is a sensitive and specific biomarker for MiT family aberration-associated renal cell carcinoma.

Microphthalmia-associated transcription factor (MiT) family aberration-associated renal cell carcinoma (MiTF-RCC) is a subtype of renal cell carcinoma harboring recurrent chromosomal rearrangements involving TFE3 or TFEB genes. MiTF-RCC is morphologically diverse, can histologically resemble common RCC subtypes like clear cell RCC and papillary RCC, and often poses a diagnostic challenge in genitourinary clinical and pathology practice. To characterize the MiTF-RCC at the molecular level and identify biomarker signatures associated with MiTF-RCC, we analyzed RNAseq data from MiTF-RCC, other RCC subtypes and benign kidney. Upon identifying TRIM63 as a cancer-specific biomarker in MiTF-RCC, we evaluated its expression independently by RNA in situ hybridization (RNA-ISH) in whole tissue sections from 177 RCC cases. We specifically included 31 cytogenetically confirmed MiTF-RCC cases and 70 RCC cases suspicious for MiTF-RCC in terms of clinical and morphological features, to evaluate and compare TRIM63 RNA-ISH results with the results from TFE3/TFEB fluorescence in situ hybridization (FISH), which is the current clinical standard. We confirmed that TRIM63 mRNA was highly expressed in all classes of MiTF-RCC compared to other renal tumor categories, where it was mostly absent to low. While the TRIM63 RNA-ISH and TFE3/TFEB FISH results were largely concordant, importantly, TRIM63 RNA-ISH was strongly positive in TFE3 FISH false-negative cases with RBM10-TFE3 inversion. In conclusion, TRIM63 can serve as a diagnostic marker to distinguish MiTF-RCC from other renal tumor subtypes with overlapping morphology. We suggest a combination of TFE3/TFEB FISH and TRIM63 RNA-ISH assays to improve the accuracy and efficiency of MiTF-RCC diagnosis. Accurate diagnosis of MiTF-RCC and other RCC subtypes would enable effective targeted therapy and avoid poor therapeutic response due to tumor misclassification.

The Lbx1 lineage differentially contributes to inhibitory cell types of the dorsal cochlear nucleus, a cerebellum-like structure, and the cerebellum.

The dorsal cochlear nucleus (DCN) is a mammalian-specific nucleus of the auditory system. Anatomically, it is classified as a cerebellum-like structure. These structures are proposed to share genetic programs with the cerebellum. Previous analyses demonstrated that inhibitory serial sister cell types (SCTs) of the DCN and cerebellum are derived from the pancreatic transcription factor 1a (Ptf1a) lineage. Postmitotic neurons of the Ptf1a lineage often express the transcription factor Ladybird homeobox protein homolog 1 (Lbx1) which is involved in neuronal cell fate determination. Lbx1 is therefore an attractive candidate for a further component of the genetic program shared between the DCN and cerebellum. Here, we used cell-type specific marker analysis in combination with an Lbx1 reporter mouse line to analyze in both tissues which cell types of the Ptf1a lineage express Lbx1. In the DCN, stellate cells and Purkinje-like cartwheel cells were part of the Lbx1 lineage and Golgi cells were not, as determined by cell counts. In contrast, in the cerebellum, stellate cells and Golgi cells were part of the Lbx1 lineage and Purkinje cells were not. Hence, two out of three phenotypically similar cell types differed with respect to their Lbx1 expression. Our study demonstrates that Lbx1 is differentially recruited to the developmental genetic program of inhibitory neurons both within a given tissue and between the DCN and cerebellum. The differential expression of Lbx1 within the DCN and the cerebellum might contribute to the genetic individuation of the inhibitory SCTs to adapt to circuit specific tasks.

Shotgun proteomics for the preliminary identification of biomarkers of beef sensory tenderness, juiciness and chewiness from plasma and muscle of young Limousin-sired bulls.

Label free shotgun proteomics was used to analyse plasma and Longissimus muscle biopsies of Limousin-sired bulls, classified as 5 high-quality and 5 low-quality meat based on sensory texture traits (tenderness, juiciness and chewiness). A total of 31 putative protein biomarkers (16 in plasma and 15 in muscle) differed significantly in abundance between the two quality groups. The proteins were associated with muscle structure, energy metabolism, heat shock proteins, oxidative stress and proteolysis related pathways. Among them, B2M, AHSG, APOA4 and HP-20 (plasma), PFKM, MYH2, PTER, GSTM1 and MYPN (muscle) were good predictors of the three texture quality traits. Further, significant correlations were identified for FETUB, SERPINA7, ASL, TREH, HP, HP-25, AZGP1, APCS and SYT15, which are novel biomarkers from plasma that warrant further evaluation. This study is a significant step forward in elucidating proteomic profiles in bovine bio-fluids and muscle tissue, which may ultimately provide opportunities to processors for early assessment of beef sensory quality.

Myofibrillar protein characteristics of fast or slow frozen pork during subsequent storage at -3 °C.

This study aimed to investigate the effect of storage at -3 °C on myofibrillar protein in fast or slow frozen pork. Five pork loins at 48 h post-mortem were subjected to either fast (cold metal plate/-80 °C) or slow freezing (still air/-20 °C) followed by storage at -3 °C for 0, 1, 3, and 7 days before thawing. Freezing rate significantly influenced myofibrillar proteins within 3 days at -3 °C, evidenced by higher thaw loss, higher surface hydrophobicity and reduced water-holding of myofibrils, and accelerated appearance of a myosin-4 fragment (160 kDa) in slow freezing. However, these observed differences disappeared after 7 days of storage at -3 °C. The meat pH after thawing did not differ between fast and slow freezing rate. However, the pH values after thawing in both groups decreased with extended storage at -3 °C. Our results suggest that the beneficial effects of fast freezing are gradually lost by holding at -3 °C due to more extensive protein denaturation.

Methods for Proteomics-Based Analysis of the Human Muscle Secretome Using an In Vitro Exercise Model.

Over the last decade, the skeletal muscle as a secretory organ has gained importance. A growing number of peptides is described which are produced and released by the muscle fibers and work in an autocrine, paracrine, and endocrine fashion. The contraction-induced secretion of these myokines is considered to contribute to the health promoting effects of exercise. To gain further insights into the molecular processes that occur during contraction, an in vitro exercise model, electric pulse stimulation (EPS), was established. Recent publications show that this model is suitable to electrostimulate human skeletal muscle cells and thus mimic muscle contraction in vitro. Here, we provide a detailed protocol for the proteomics-based analysis of the human muscle secretome, starting with the cultivation of human myotubes and ending with sample preparation for targeted and untargeted proteome analysis of the cell culture supernatant. This workflow should allow for deeper insights into the complex nature of the muscle secretome and the identification of new myokines which might help to understand the cross talk of the working muscle with different organs and the beneficial effects of exercise.

Metabolic fingerprinting of in-bag dry- and wet-aged lamb with rapid evaporative ionisation mass spectroscopy.

The effect of in-bag dry- and wet-ageing on metabolite profiles of lamb legs was determined using Rapid Evaporative Ionisation Mass Spectrometry (REIMS). Using orthogonal projection to latent structures-discriminant analysis (OPLS-DA) with REIMS, 1705 metabolite ions were identified (Q2 = 0.86) in four muscles: m. semimembranosus, m. biceps femoris, m. vastus lateralis and m. rectus femoris. A total of 663 metabolites differed between ageing methods (P < 0.05) which mainly resulted from proteolysis and lipid metabolism. Dry-aged lamb had higher pH (P = 0.016) and lower moisture content (P = 0.034) than the wet-aged. Dry-ageing produced more (P < 0.05) smaller sized metabolites including dipeptides and free amino acids and lipid oxidation metabolites compared to wet-aged equivalents. Different muscles had distinct REIMS metabolic profiles. Outcomes of this study demonstrated that REIMS can be used for authentication between in-bag dry- and wet-aged lamb based on their metabolic fingerprints.

Jasplakinolide Attenuates Cell Migration by Impeding Alpha-1-syntrophin Protein Phosphorylation in Breast Cancer Cells.

BACKGROUND: Alpha-1-syntrophin (SNTA1) is emerging as a novel modulator of the actin cytoskeleton. SNTA1 binds to F-actin and regulates intracellular localization and activity of various actin organizing signaling molecules. Aberration in syntrophin signaling has been closely linked with deregulated growth connected to tumor development/metastasis and its abnormal over expression has been observed in breast cancer. In the present work the effect of jasplakinolide, an actin-binding cyclodepsipeptide, on the SNTA1 protein activity and SNTA1 mediated downstream cellular events was studied in MDA-MB-231 breast cancer cell line. METHODS: SNTA1 protein levels and phosphorylation status were determined in MDA-MB-231 cells post jasplakinolide exposure using western blotting and immunoprecipitation techniques respectively. MDA-MB-231 cells were transfected with WT SNTA1 and DM SNTA1 (Y215/229 phospho mutant) and simultaneously treated with jasplakinolide. The effect of jasplakinolide and SNTA1 protein on cell migration was determined using the boyden chamber assay. RESULTS: Jasplakinolide treatment decreases proliferation of MDA-MB-231 cells in both dose and time dependent manner. Results suggest that subtoxic doses of jasplakinolide induce morphological changes in MDA-MB-231 cells from flat spindle shape adherent cells to round weakly adherent forms. Mechanistically, jasplakinolide treatment was found to decrease SNTA1 protein levels and its tyrosine phosphorylation status. Moreover, migratory potential of jasplakinolide treated cells was significantly inhibited in comparison to control cells. CONCLUSION: Our results demonstrate that jasplakinolide inhibits cell migration by impairing SNTA1 functioning in breast cancer cells.