New Solutions for Old Problems: How Reproductive Tissue Engineering Has Been Revolutionizing Reproductive Medicine.

Acquired disorders and congenital defects of the male and female reproductive systems can have profound impacts on patients, causing sexual and endocrine dysfunction and infertility, as well as psychosocial consequences that affect their self-esteem, identity, sexuality, and relationships. Reproductive tissue engineering (REPROTEN) is a promising approach to restore fertility and improve the quality of life of patients with reproductive disorders by developing, replacing, or regenerating cells, tissues, and organs from the reproductive and urinary systems. In this review, we explore the latest advancements in REPROTEN techniques and their applications for addressing degenerative conditions in male and female reproductive organs. We discuss current research and clinical outcomes and highlight the potential of 3D constructs utilizing biomaterials such as scaffolds, cells, and biologically active molecules. Our review offers a comprehensive guide for researchers and clinicians, providing insights into how to reestablish reproductive tissue structure and function using innovative surgical approaches and biomaterials. We highlight the benefits of REPROTEN for patients, including preservation of fertility and hormonal production, reconstruction of uterine and cervical structures, and restoration of sexual and urinary functions. Despite significant progress, REPROTEN still faces ethical and technical challenges that need to be addressed. Our review underscores the importance of continued research in this field to advance the development of effective and safe REPROTEN approaches for patients with reproductive disorders.

Estetrol Increases Progesterone Genetic Response without Triggering Common Estrogenic Effects in Endometriotic Cell Lines and Primary Cultures.

Estetrol (E4), a natural estrogen produced by the human fetal liver, is actively studied for menopause and breast cancer treatment. It has low side effects and preferential estrogen receptor alpha (ERα) affinity. There are no data about its effects on endometriosis, a common gynecological disease affecting 6-10% of cycling women, generating painful pelvic lesions and infertility. Current combined hormone treatment (progestins and estrogens) is safe and efficient; nevertheless, one-third of patients develop progesterone (P4) resistance and recurrence by reducing P4 receptors (PRs) levels. We aimed to compare E4 and 17ß-estradiol (E2) effects using two human endometriotic cell lines (epithelial 11Z and stromal Hs832 cells) and primary cultures from endometriotic patients. We evaluated cell growth (MTS), migration (wound assay), hormone receptors levels (Western blot), and P4 response by PCR array. Compared to E2, E4 did not affect cell growth or migration but increased estrogen receptor alpha (ERα) and PRs, and reduced ERß. Finally, the incubation with E4 improved the P4 gene response. In conclusion, E4 increased PRs levels and genetic response without inducing cell growth or migration. These results suggest that E4 might be useful for endometriosis treatment avoiding P4 resistance; however, evaluating its response in more complex models is required.

Pediatric Swine Model of Methicillin-Resistant Staphylococcus aureus Sepsis-Induced Coagulopathy, Disseminated Microvascular Thrombosis, and Organ Injuries.

Sepsis-induced coagulopathy leading to disseminated microvascular thrombosis is associated with high mortality and has no existing therapy. Despite the high prevalence of Gram-positive bacterial sepsis, especially methicillin-resistant Staphylococcus aureus (MRSA), there is a paucity of published Gram-positive pediatric sepsis models. Large animal models replicating sepsis-induced coagulopathy are needed to test new therapeutics before human clinical trials. HYPOTHESIS: Our objective is to develop a pediatric sepsis-induced coagulopathy swine model that last 70 hours. METHODS AND MODELS: Ten 3 weeks old piglets, implanted with telemetry devices for continuous hemodynamic monitoring, were IV injected with MRSA (n = 6) (USA300, Texas Children's Hospital 1516 strain) at 1 × 109 colony forming units/kg or saline (n = 4). Fluid resuscitation was given for heart rate greater than 50% or mean arterial blood pressure less than 30% from baseline. Acetaminophen and dextrose were provided as indicated. Point-of-care complete blood count, prothrombin time (PT), activated thromboplastin time, d-dimer, fibrinogen, and specialized coagulation assays were performed at pre- and post-injection, at 0, 24, 48, 60, and 70 hours. Piglets were euthanized and necropsies performed. RESULTS: Compared with the saline treated piglets (control), the septic piglets within 24 hours had significantly lower neurologic and respiratory scores. Over time, PT, d-dimer, and fibrinogen increased, while platelet counts and activities of factors V, VII, protein C, antithrombin, and a disintegrin and metalloproteinase with thrombospondin-1 motifs (13th member of the family) (ADAMTS-13) decreased significantly in septic piglets compared with control. Histopathologic examination showed minor focal organ injuries including microvascular thrombi and necrosis in the kidney and liver of septic piglets. INTERPRETATIONS AND CONCLUSIONS: We established a 70-hour swine model of MRSA sepsis-induced coagulopathy with signs of consumptive coagulopathy, disseminated microvascular thrombosis, and early organ injuries with histological minor focal organ injuries. This model is clinically relevant to pediatric sepsis and can be used to study dysregulated host immune response and coagulopathy to infection, identify potential early biomarkers, and to test new therapeutics.

Molecular Characterization of Embryos with Different Buoyancy Levels in the Yellowtail Kingfish (Seriola lalandi).

The buoyancy of eggs and embryos is associated with successful development in pelagic fish. Buoyancy is the result of oocyte hydration, which depends on the osmotic force exerted by free amino acids (FAA) generated by yolk proteolysis, and cathepsins are the main enzymes involved in this process. Seriola lalandi is a pelagic fish whose farming has been hampered by development failure that have been partially attributed to decreased buoyancy of embryos. Therefore, the aim of this study was to compare the mRNA expression and activity of cathepsins B, D, and L, as well as the FAA content in floating and low-floating embryos at different developmental stages. The chosen stages were eggs, morula, blastula, gastrula and 24 h embryos. Complementary assessments showed that there were no differences attributed to buoyancy status in embryo and oil droplet diameters, as well as the transcriptional status at any developmental stage. Cathepsin B did not show differences in mRNA expression or activity related to buoyancy at any stage. Cathepsin D displayed higher transcript and activity levels only in low-floating eggs compared with those floating. Cathepsin L showed higher expression in floating eggs and 24 h embryos compared with that of low-floating, but the activity of this enzyme was higher in floating eggs and morula. Total FAA content constantly decreased throughout development in floating embryos, but it was always higher than low-floating embryos until gastrula stage. In 24 h embryos floating and low-floating embryos share similar quantities of FAA. In summary, differences in the expression and activity of cathepsins between floating and low-floating embryos could be revealed at specific embryonic stages, suggesting different functions of these enzymes throughout development. Besides 24 h embryos, FAA content seems to be a decisive factor for buoyancy of embryos during early development of S. lalandi. Overall, considering the main role of cathepsins and FAA in buoyancy acquisition process and therefore in both embryo quality and viability, our study identifies good marker candidates to evaluate embryo quality in the farming of this species.

Pediatric Critical Care Transport: Survey of Current State in Latin America. Latin American Society of Pediatric Intensive Care Transport Committee.

METHODS: An electronic, anonymous, multicenter survey housed by Monkey Survey was sent to physicians in LA and included questions about hospital and pediatric critical transport, resources available and level of car. Nineteen Latin-American countries were asked to complete the survey. RESULTS: A total of 212 surveys were analyzed, achieving a representativity of 19 LA countries, being most participants (59.4%, n = 126) from South America (Argentina, Bolivia, Brazil, Chile, Colombia, Ecuador, Paraguay, Peru, Uruguay and Venezuela). Most surveys were conducted by physicians of tertiary level centers (60.8%, n = 129), most of the institutions were classified by the participants as public health care centers (81.6%, n = 173). Most of the surveyed physicians (63.7%, n = 135) reported that there is a coordination center for critical care transport (CCT). In most cases, physicians report that a unified transport system for pediatric critical patients does not exist in their countries (67.45%, n = 143). Only 59 (30.7%) surveys reported the use of an exclusively pediatric critical care transport system. Most of these transport systems are described as a mixture of public and private efforts (51.56%, n = 99), but there is also a considerable involvement of government-funded critical transport systems (43.75%, n = 84). Specific training for personnel devoted to transportation of critically ill patients is reported in 55.6% (90), and the medical equipment necessary to carry out the transport is available in 67.7%. The majority (83.95%, n = 136) mentioned that access to advanced life support courses is possible. Training in triage and disaster is available in 44.1%. Physicians and registered nurse were identified as the transport providers in 41.5%, and only one third were made by pediatricians-pediatric nurse. The main reasons for transfers were respiratory illness, neonatal pathologies, trauma, infectious diseases, and neurological conditions. Overall, pediatric transport was reported as insufficient (70.19%, n = 148) by the surveyed physicians in LA and nonexisting by some of them (6.83%, n = 15). There were no regulations or laws in the majority of the surveyed countries (63.13%), and in the places where physicians reported regulatory laws, there were no dissemination (84.9%) by the local authorities. CONCLUSIONS: In LA, there is a great variability in personnel training, equipment for pediatric-neonatal transport, transport team composition, and characterization of critical care transport systems. Continued efforts to improve conditions in our countries by generating documents that standardize practices and generating scientific information on the epidemiology of pediatric transfers, especially of critically ill patients, may help reduce patient morbidity and mortality.

Embryo Buoyancy and Cell Death Gene Expression During Embryogenesis of Yellow-Tail Kingfish Seriola lalandi.

In pelagic fish, embryo buoyancy is a noteworthy aspect of the reproductive strategy, and is associated with overall quality, survival, and further developmental success. In captivity, the loss of buoyancy of early embryos correlates with high mortality that might be related to massive cell death. Therefore, the aim of this study was to evaluate under captivity conditions the expression of genes related to the apoptosis process during the early embryonic development of the pelagic fish Seriola lalandi, and its relationship to the buoyancy of embryos. The relative expression of bcl2, bax-like, casp9, casp8, and casp3 was evaluated by RT-qPCR and FasL/Fas protein levels by western blot in five development stages of embryos sorted as floating or low-floating. All genes examined were expressed in both floating and low-floating embryos up to 24 h of development. Expression of the pro-apoptotic factors bax, casp9, casp8, and casp3 was higher in low-floating as compared with floating embryos in a developmental stage-specific manner. In contrast, there was no difference in expression of bcl2 between floating and low-floating embryos. Fas protein was detected as a single band in floating embryos without changes in expression throughout development; however, in low-floating embryos, three higher intensity reactive bands were detected in the 24-h embryos. Interestingly, FasL was only detected at 24-h in floating embryos, whereas in low-floating samples this ligand was present at all stages, with a sharp increase as development progressed. Cell death, as evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, was highly increased in low-floating embryos as compared to floating embryos throughout all developmental stages, with the highest levels observed during the gastrula stage and at 24 h. The results of this study suggest that an increase in cell death, probably associated with the intrinsic and extrinsic apoptosis pathways, is present in low-floating embryos that might explain their lower developmental potential under captivity conditions.

A decrease of docosahexaenoic acid in testes of mice fed a high-fat diet is associated with impaired sperm acrosome reaction and fertility.

Obesity is a major worldwide health problem that is related to most chronic diseases, including male infertility. Owing to its wide impact on health, mechanisms underlying obesity-related infertility remain unknown. In this study, we report that mice fed a high-fat diet (HFD) for over 2 months showed reduced fertility rates and increased germ cell apoptosis, seminiferous tubule degeneration, and decreased intratesticular estradiol (E2) and E2-to-testosterone ratio. Interestingly, we also detected a decrease in testicular fatty acid levels, behenic acid (C22:0), and docosahexaenoic acid (DHA, 22:6n-3), which may be related to the production of dysfunctional spermatozoa. Overall, we did not detect any changes in the frequency of seminiferous tubule stages, sperm count, or rate of in vitro capacitation. However, there was an increase in spontaneous and progesterone-induced acrosomal exocytosis (acrosome reaction) in spermatozoa from HFD-fed mice. These data suggest that a decrease in E2 and fatty acid levels influences spermatogenesis and some steps of acrosome biogenesis that will have consequences for fertilization. Thus, our results add new evidence about the adverse effect of obesity in male reproduction and suggest that the acrosomal reaction can also be affected under this condition.

Daily exposure to phthalates and alkylphenols alters miR biogenesis and expression in mice ovaries.

Reproductive hormone imbalance in infertile women is correlated to high levels of phthalates and alkylphenols, which are among endocrine-disrupting chemicals (EDCs). Previous studies have shown that they interfere with gene expression by deregulating levels of microRNAs (miRs), small non-coding RNAs targeting mRNAs encoding enzymes in the hormone biosynthesis pathway. However, this effect depends on the target organ, dose and whether or not they are alone or in mixtures. Our goal was to study whether the biosynthesis, and a specific group of miRs targeting mRNAs encoding enzymes in steroid hormone biosynthesis, are deregulated in the ovaries of female mice chronically exposed to a mixture of three phthalates (DEHP+DBP+BBP) and two alkylphenols (NP+OP) at a human environmentally relevant dose. We performed qPCR and Western blot assays along with a bioinformatics approach and found that this mixture modified the biogenesis machinery of miRs, inducing an increase in the mRNA levels of Drosha and Dicer1 and DROSHA protein levels. In addition, we found changes in the precursor and mature forms of miR-96-5p, miR-200b-3p, miR-365-3p, miR-378a-3p and miR-503-5p which target steroidogenic pathway enzymes. Finally, using primary granulosa cell culture, we confirmed that miR-200b-3p targets Cyp19a1, transcript encoding CYP19A1, the enzyme that produces estradiol (E2). These results indicate that chronic exposure to phthalates and alkylphenols mixture alters the biogenesis of ovary miRs and increases the expression of miRs implicated in the control of steroidal hormone synthesis in female mice, thus contributing to reproductive pathologies.

Derangement of Arginine and Related Amino Acids in Children Undergoing Surgery for Congenital Heart Disease With Cardiopulmonary Bypass.

Arginine is a conditionally essential amino acid, the precursor for nitric oxide and a key factor in cell proliferation, protein synthesis, and energy metabolism. When there is increased demand in the setting of inflammation, ischemia-reperfusion injury, and organ dysfunction, endogenous arginine production falls short, and external supplementation may be necessary. The goal of this study was to assess changes in concentrations of plasma arginine, citrulline, ornithine, glutamine, and plasma arginase in infants and children undergoing surgery for congenital heart disease with cardiopulmonary bypass. DESIGN: Prospective observational study. SETTING: The study was conducted in the Heart Center at Texas Children's Hospital. SUBJECTS: Children undergoing surgery for congenital heart disease with cardiopulmonary bypass. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Serial perioperative blood samples were collected for quantification of amino acids, arginase, nitric oxide metabolites, and markers of organ function (lactate, Pao2/Fio2 ratio, and creatinine clearance). Thirty children (18 males) were included in the study; median (interquartile range) age 0.5 years (0.3-0.9 yr). The mean ± sd for plasma amino acid concentrations before cardiopulmonary bypass: arginine 62 ± 20 µmol/L, citrulline 24 ± 6 µmol/L, ornithine 53 ± 32 µmol/L, and glutamine 591 ± 126 µmol/L. Arginine concentration was decreased within the first 24 hours (43 ± 15 µmol/L; p = 0.004), citrulline and glutamine concentrations decreased over the first 48 hours (11 ± 4 µmol/L; p < 0.001 and 493 ± 131 µmol/L; p = 0.019, respectively) and were associated with an increase in arginase (3.8 ± 3 µg/mL; p < 0.05). There was an increase in Vasoactive-Inotropic Score (5.9 ± 19 vs 0.5 ± 2; p < 0.001), decrease in creatinine clearance (76 ± 24 vs 93 ± 31; p = 0.002), and Pao2/Fio2 ratio (243 ± 138 vs 374 ± 200; p = 0.007) comparing to baseline. CONCLUSIONS: A widely variable degree of arginine, citrulline, and glutamine depletion occurs in children after surgery for congenital heart disease. These findings were associated with increased arginase and coincide with some of the markers of organ perfusion.

Antigen retrieval by citrate solution improves western blot signal.

In the present work, we describe and evaluate an additional step to the standard western blot protocol to increase signal strength after revealing. Weak or absence of signal is a common issue in western blot protocol leading to unexpected results. In our Antigen Retrieval for Western Blot Method (ARWB method), after transfer, the membrane was incubated in a citrate buffer following normal antigen retrieval procedure used for immunohistochemistry. Later, standard protocol was performed in order to reveal and compare with unexposed membranes to this antigen retrieval step. Signal in bands obtained by the modified protocol resulted significantly higher (in all 13 antibodies analyzed) compared to standard protocol. Some bands were only visible after citrate incubation. This method is a simple and economical way to improve results in western blot analysis. •The ARWB method significantly increases band's density in all antibodies analyzed.•Protein localization does not influence the efficacy of the ARWB method since membrane and citoplasmatic proteins bands increase their signal in a similar way after the protocol is performed.•This ARWB method is simple, safe, economical and undoubtedly helpful in immunoblotting for proteins with weak signal.

Intermittent bolus feeding promotes greater lean growth than continuous feeding in a neonatal piglet model.

Background: Orogastric tube feeding is indicated in neonates with an impaired ability to ingest food normally and can be administered with an intermittent bolus or continuous feeding schedule. Objectives: The objectives were to 1) compare the long-term effect of continuous with intermittent feeding on growth using the newborn pig as a model, 2) determine whether feeding frequency alters lean tissue and fat mass gain, and 3) identify the signaling mechanisms by which protein deposition is controlled in skeletal muscle in response to feeding frequency. Design: Neonatal pigs were fed the same amount of a balanced formula by orogastric tube either as an intermittent bolus meal every 4 h (INT) or as a continuous infusion (CON). Body composition was assessed at the start and end of the study by dual-energy X-ray absorptiometry, and hormone and substrate profiles, muscle mass, protein synthesis, and indexes of nutrient and insulin signaling were measured after 21 d. Results: Body weight, lean mass, spine length, and skeletal muscle mass were greater in the INT group than in the CON group. Skeletal muscle fractional protein synthesis rates were greater in the INT group after a meal than in the CON group and were associated with higher circulating branched-chain amino acid and insulin concentrations. Skeletal muscle protein kinase B (PKB) and ribosomal protein S6 kinase phosphorylation and eukaryotic initiation factor (eIF) 4E-eIF4G complex formation were higher, whereas eIF2α phosphorylation was lower in the INT group than in the CON group, indicating enhanced activation of insulin and amino acid signaling to translation initiation. Conclusions: These results suggest that when neonates are fed the same amounts of nutrients as intermittent meals rather than continuously there is greater lean growth. This response can be ascribed, in part, to the pulsatile pattern of amino acids, insulin, or both induced by INT, which enables the responsiveness of anabolic pathways to feeding to be sustained chronically in skeletal muscle.

Important role of collective cell migration and nerve fiber density in the development of deep nodular endometriosis.

OBJECTIVE: To evaluate deep nodular endometriotic lesions induced in baboons over 12 months and analyze collective cell migration and nerve fiber density. DESIGN: Morphologic and immunohistochemical analysis of endometriotic lesions induced in baboons over the course of 1 year. SETTING: Academic research unit. ANIMAL(S): Three female baboons (Papio anubis). INTERVENTION(S): Recovery of induced deep nodular endometriotic nodules from baboons. MAIN OUTCOME MEASURE(S): Evaluation of the morphology of glands by analysis of the center of lesions and the invasion front; immunohistochemical staining with Ki67, E-cadherin, and ß-catenin for investigation of mitotic activity and cell-cell junctions, and with protein gene product 9.5 and nerve growth factor (NGF) for study of nerve fiber density (NFD). RESULT(S): All (100%) of the lesions were invasive 1 year after induction, compared with 42.29% after 6 months. Glands from the invasion front showed significantly reduced thickness but significantly higher mitotic activity. E-Cadherin and ß-catenin expression were similar between the center and front. NFD was significantly higher in lesions induced after 1 year than after 6 months, and NGF expression was significantly lower in 1-year lesions than in 6-month lesions. CONCLUSION(S): Nodular endometriotic lesions induced in the baboon model were found to be significantly more invasive and innervated after 12 months than after 6 months. The invasive phenotype was highly expressed in glands at the invasion front, and our study suggests that nerve fibers play a role in the development of lesions as observed in women.

Diabetic concentrations of metformin inhibit platelet-mediated ovarian cancer cell progression.

Clinical studies have suggested a survival benefit in ovarian cancer patients with type 2 diabetes mellitus taking metformin, however the mechanism by which diabetic concentrations of metformin could deliver this effect is still poorly understood. Platelets not only represent an important reservoir of growth factors and angiogenic regulators, they are also known to participate in the tumor microenvironment implicated in tumor growth and dissemination. Herein, we investigated if diabetic concentrations of metformin could impinge upon the previously reported observation that platelet induces an increase in the tube forming capacity of endothelial cells (angiogenesis) and upon ovarian cancer cell aggressiveness. We demonstrate that metformin inhibits the increase in angiogenesis brought about by platelets in a mechanism that did not alter endothelial cell migration. In ovarian cancer cell lines and primary cultured cancer cells isolated from the ascitic fluid of ovarian cancer patients, we assessed the effect of combinations of platelets and metformin upon angiogenesis, migration, invasion and cancer sphere formation. The enhancement of each of these parameters by platelets was abrogated by the present of metformin in the vast majority of cancer cell cultures tested. Neither metformin nor platelets altered proliferation; however, metformin inhibited the increase in phosphorylation of focal adhesion kinase induced by platelets. We present the first evidence suggesting that concentrations of metformin present in diabetic patients may reduce the actions of platelets upon both endothelial cells and cancer cell survival and dissemination.

Gynecological and obstetrical outcomes after laparoscopic repair of a cesarean scar defect in a series of 38 women.

OBJECTIVE: To evaluate gynecological and obstetrical outcomes, as well as remaining myometrial thickness, after laparoscopic repair of a cesarean scar. DESIGN: Observational study and prospective evaluation of the remaining myometrium before and after repair. SETTING: Academic department in a university hospital. PATIENT(S): A series of 38 symptomatic women with cesarean scar defects and remaining myometrial thickness of less than 3 mm, according to magnetic resonance imaging. INTERVENTION(S): Laparoscopic repair of the defect. MAIN OUTCOMES MEASURE(S): Increase in myometrial thickness at the site of cesarean section, gynecological and obstetrical outcomes, and histological analysis of the defect after excision. RESULT(S): The mean thickness of the myometrium increased significantly from 1.43 ± 0.7 mm before surgery to 9.62 ± 1.8 mm after surgery. All but three patients were free of symptoms. Among the 18 women with infertility, eight (44%) became pregnant and delivered healthy babies by cesarean section at 38-39 weeks of gestation. Histological analysis, performed in all 38 cases, revealed the presence of endometriosis in eight women (21.1%). Muscle fiber density was significantly lower compared with adjacent myometrium. CONCLUSION(S): In symptomatic women with residual myometrial thickness of less than 3 mm who wish to conceive, laparoscopic repair could be considered an appropriate approach.

Leucine supplementation stimulates protein synthesis and reduces degradation signal activation in muscle of newborn pigs during acute endotoxemia.

Sepsis disrupts skeletal muscle proteostasis and mitigates the anabolic response to leucine (Leu) in muscle of mature animals. We have shown that Leu stimulates muscle protein synthesis (PS) in healthy neonatal piglets. To determine if supplemental Leu can stimulate PS and reduce protein degradation (PD) signaling in neonatal muscle during endotoxemia, overnight-fasted neonatal pigs were infused for 8 h with LPS or saline while plasma amino acids, glucose, and insulin were maintained at fasting levels during pancreatic-substrate clamps. Leu or saline was infused during the last hour. Markers of PS and PD were determined in skeletal muscle. Compared with controls, Leu increased PS in longissimus dorsi (LD), gastrocnemius, and soleus muscles. LPS decreased PS in these three muscles by 36%, 28%, and 38%, but Leu antagonized that reduction by increasing PS by 84%, 81%, and 83%, respectively, when supplemented to LPS. Leu increased eukaryotic translation initiation factor (eIF)3b-raptor interactions, eIF4E-binding protein-1, and S6 kinase 1 phosphorylation as well as eIF4E·eIF4G complex formation in LD, gastrocnemius, and soleus muscles of control and LPS-treated pigs. In LD muscle, LPS increased the light chain (LC)3-II-to-LC3 ratio and muscle-specific RING finger (MuRF-1) abundance but not atrogin-1 abundance or AMP-activated protein kinase-α phosphorylation. Leu supplementation to LPS-treated pigs reduced the LC3-II-to-LC3 ratio, MuRF-1 abundance, and AMP-activated protein kinase-α phosphorylation compared with LPS alone. In conclusion, parenteral Leu supplementation attenuates the LPS-induced reduction in PS by stimulating mammalian target of rapamycin complex 1-dependent translation and may reduce PD by attenuating autophagy-lysosome and MuRF-1 signaling in neonatal skeletal muscle.