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Ludmila Pierdevara,1 Ana María Porcel-Gálvez,2 Alexandra Maria Ferreira da Silva,1 Sérgio Barrientos Trigo,3 Margarida Eiras4 1Escuela Internacional de Doctorado, Universidad de Sevilla, Sevilla, Spain; 2Nursing Department, Escuela Internacional de Doctorado, University of Seville, Sevilla, Spain; 3Department of Nursing, Escuela Internacional de Doctorado, University of Seville, Sevilla, Spain; 4H&RC ESTeSL-IPL, Lisbon, PortugalCorrespondence: Ludmila Pierdevara Praça do Poder Local, Lote 7ª, 6º Frente, Lagos 8600-524, PortugalTel +351 963605271Email pierdevara@hotmail.comPurpose: To adapt and validate the Global Trigger Tool (IHI-GTT), which identifies and analyzes adverse events (AE) in hospitalized patients and their measurement properties in the Portuguese context.Methods: A retrospective cross-sectional study was based on a random sample of 90 medical records. The stages of translation and cross-cultural adaptation of the IHI-GTT were based on the Cross-Cultural Adaptation Protocol that originated from the Portuguese version, GTT-PT, for the hospital context in medical-surgical departments. Internal consistency, reliability, reproducibility, diagnostic tests, and discriminatory predictive value were investigated.Results: The final phase of the GTT-PT showed insignificant inconsistencies. The pre-test phase confirmed translation accuracy, easy administration, effectiveness in identifying AEs, and relevance of integrating it into hospital risk management. It had a sensitivity of 97.8% and specificity of 74.8%, with a cutoff point of 0.5, an accuracy of 83%, and a positive predictive value of 69.8% and a negative predictive value of 0.98%.Conclusion: The GTT-PT is a reliable, accurate, and valid tool to identify AE, with robust measurement properties.Keywords: medical errors, patient safety, risk management

Background: Understanding how urban environments influence people's health, especially as individuals age, can help identify ways to improve health in the rapidly urbanizing and rapidly aging populations., Objectives: To investigate the association between age and self-reported health (SRH) in adults living in Latin-American cities and whether gender and city-level socioeconomic characteristics modify this association., Methods: Cross-sectional analyses of 71,541 adults aged 25-97 years, from 114 cities in 6 countries (Argentina, Brazil, Colombia, Chile, El Salvador, and Guatemala), as part of the Salud Urbana en America Latina (SALURBAL) Project. We used individual-level age, gender, education, and self-reported health (SRH) data from harmonized health surveys. As proxies for socioeconomic environment we used a city-level socioeconomic index (SEI) calculated from census data, and gross domestic product (GDP) per-capita. Multilevel Poisson models with a robust variance were used to estimate relative risks (RR), with individuals nested in cities and binary SRH (poor SHR vs. good SRH) as the outcome. We examined effect modification by gender and city-level socioeconomic indicators., Results: Overall, 31.4% of the sample reported poor SRH. After adjusting for individual-level education, men had a lower risk of poor SRH (RR = 0.76; CI 0.73-0.78) compared to women, and gender modified the association between age and poor SRH (p-value of interaction < 0.001). In gender stratified models, the association between older age and poor SRH was more pronounced in men than in women, and in those aged 25-65 than among those 65+ (RR/10 years = 1.38 vs. 1.10 for men, and RR/10 years = 1.29 vs. 1.02 for women). Living in cities with higher SEI or higher GDP per-capita was associated with a lower risk of poor SRH. GDP per-capita modified the association between age (25-65) and SRH in men and women, with SEI the interaction was less clear., Conclusions: Across cities in Latin America, aging impact on health is significant among middle-aged adults, and among men. In both genders, cities with lower SEI or lower GDP per-capita were associated with poor SRH. More research is needed to better understand gender inequalities and how city socioeconomic environments, represented by different indicators, modify exposures and vulnerabilities associated with aging., (© 2022. The Author(s).)

Increased intracellular iron spurs mitochondrial biogenesis and respiration to satisfy high-energy demand during osteoclast differentiation and bone-resorbing activities. Transferrin receptor 1 (Tfr1) mediates cellular iron uptake through endocytosis of iron-loaded transferrin, and its expression increases during osteoclast differentiation. Nonetheless, the precise functions of Tfr1 and Tfr1-mediated iron uptake in osteoclast biology and skeletal homeostasis remain incompletely understood. To investigate the role of Tfr1 in osteoclast lineage cells in vivo and in vitro, we crossed Tfrc (encoding Tfr1)-floxed mice with Lyz2 (LysM)- Cre and Cathepsin K ( Ctsk )-Cre mice to generate Tfrc conditional knockout mice in myeloid osteoclast precursors (Tfr1 ΔLysM ) or differentiated osteoclasts (Tfr1 ΔCtsk ), respectively. Skeletal phenotyping by µCT and histology unveiled a significant increase in trabecular bone mass with normal osteoclast number in long bones of 10-week-old young and 6-month-old adult female but not male Tfr1 ΔLysM mice. Although high trabecular bone volume in long bones was observed in both male and female Tfr1 ΔCtsk mice, this phenotype was more pronounced in female knockout mice. Consistent with this gender-dependent phenomena, estrogen deficiency induced by ovariectomy decreased trabecular bone mass in Tfr1 ΔLysM mice. Mechanistically, disruption of Tfr1 expression attenuated mitochondrial metabolism and cytoskeletal organization in mature osteoclasts in vitro by attenuating mitochondrial respiration and activation of the Src-Rac1-WAVE regulatory complex axis, respectively, leading to decreased bone resorption with little impact on osteoclast differentiation. These results indicate that Tfr1-mediated iron uptake is specifically required for osteoclast function and is indispensable for bone remodeling in a gender-dependent manner., Competing Interests: BD, LW, TF, JZ, NA, KK, RL, SM, RE, MJ, XW, JF, TB, JG, AK, LG, WX, HZ No competing interests declared, SM Reviewing editor, eLife

Supplemental Digital Content is available in the text., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association.)

Osteoclasts are multinucleated cells of the monocyte/macrophage lineage that degrade bone. Here, we used lineage tracing studies-labelling cells expressing Cx3cr1, Csf1r or Flt3-to identify the precursors of osteoclasts in mice. We identified an erythromyeloid progenitor (EMP)-derived osteoclast precursor population. Yolk-sac macrophages of EMP origin produced neonatal osteoclasts that can create a space for postnatal bone marrow haematopoiesis. Furthermore, EMPs gave rise to long-lasting osteoclast precursors that contributed to postnatal bone remodelling in both physiological and pathological settings. Our single-cell RNA-sequencing data showed that EMP-derived osteoclast precursors arose independently of the haematopoietic stem cell (HSC) lineage and the data from fate tracking of EMP and HSC lineages indicated the possibility of cell-cell fusion between these two lineages. Cx3cr1 + yolk-sac macrophage descendants resided in the adult spleen, and parabiosis experiments showed that these cells migrated through the bloodstream to the remodelled bone after injury.

Objective: To analyze the adolescent motherhood trend and associated factors in under-100 000-inhabitants communities., Materials and Methods: Cross-sectional analysis of 16 686 women in under-100 000-inhabitants communities in Encuesta Nacional de Salud y Nutrición (Ensanut) 2006, 2012 and 100k 2018. We adjusted robust Poisson models with adolescent motherhood as dependent variable for women aged 12-19 and 20-24., Results: Attending school and using modern contraceptives decrease adolescent motherhood prevalence in both age groups. Among adolescent girls, having a health financing scheme, and early sexual debut in the case of adults, is positively associated with adolescent motherhood., Conclusions: It is necessary to strengthen public policies seeking to modify structural factors that provide life choices, and to maintain and strengthen the actions and coverage proposed by Estrategia Nacional para la Prevención del Embarazo en Adolescentes (ENAPEA) targeting this population., Competing Interests: Declaration of conflict of interests. The authors declare that they have no conflict of interests.

β-catenin protein needs to be precisely regulated for effective fracture repair. The pace of fracture healing slows with age, associated with a transient increase in β-catenin during the initial phase of the repair process. Here we examined the ability of pharmacologic agents that target β-catenin to improve the quality of fracture repair in old mice. 20 month old mice were treated with Nefopam or the tankyrase inhibitor XAV939 after a tibia fracture. Fractures were examined 21 days later by micro-CT and histology, and 28 days later using mechanical testing. Daily treatment with Nefopam for three or seven days but not ten days improved the amount of bone present at the fracture site, inhibited β-catenin protein level, and increased colony forming units osteoblastic from bone marrow cells. At 28 days, treatment increased the work to fracture of the injured tibia. XAV939 had a more modest effect on β-catenin protein, colony forming units osteoblastic, and the amount of bone at the fracture site. This data supports the notion that high levels of β-catenin in the early phase of fracture healing in old animals slows osteogenesis, and suggests a pharmacologic approach that targets β-catenin to improve fracture repair in the elderly.

The pace of repair declines with age and, while exposure to a young circulation can rejuvenate fracture repair, the cell types and factors responsible for rejuvenation are unknown. Here we report that young macrophage cells produce factors that promote osteoblast differentiation of old bone marrow stromal cells. Heterochronic parabiosis exploiting young mice in which macrophages can be depleted and fractionated bone marrow transplantation experiments show that young macrophages rejuvenate fracture repair, and old macrophage cells slow healing in young mice. Proteomic analysis of the secretomes identify differential proteins secreted between old and young macrophages, such as low-density lipoprotein receptor-related protein 1 (Lrp1). Lrp1 is produced by young cells, and depleting Lrp1 abrogates the ability to rejuvenate fracture repair, while treating old mice with recombinant Lrp1 improves fracture healing. Macrophages and proteins they secrete orchestrate the fracture repair process, and young cells produce proteins that rejuvenate fracture repair in mice.

The dynorphin (DYN) peptide family includes opioid and non-opioid peptides, yet the physiological role of the non-opioid DYN peptides remains poorly understood. Recent evidence shows that administering the non-opioid peptide DYN-A 2-17 into the paraventricular hypothalamic nucleus (PVN) simultaneously increased short-term intake of standard rodent chow and spontaneous physical activity (SPA). The present studies aimed to expand upon the mechanisms and role of DYN-A 2-17 on food intake and energy expenditure. Injection of DYN-A 2-17 in PVN increased SPA, energy expenditure and wheel running in the absence of food. Repeated DYN-A 2-17 injection in PVN increased short-term chow intake, but this effect habituated over time and failed to alter cumulative food intake, body weight or adiposity. Pre-treatment with a CRF receptor antagonist into PVN blocked the effects of DYN-A 2-17 on food intake while injection of DYN-A 2-17 in PVN increased plasma ACTH. Finally, as DYN peptides are co-released with orexin peptides, we compared the effects of DYN-A 2-17 to orexin-A and the opioid peptide DYN-A 1-13 on food choice and intake in PVN when palatable snacks and chow were available. DYN-A 1-13 selectively increased intake of palatable snacks. DYN-A 2-17 and orexin-A decreased palatable snack intake while orexin-A also increased chow intake. These findings demonstrate that the non-opioid peptide DYN-A 2-17 acutely regulates physical activity, energy expenditure and food intake without long-term effects on energy balance. These data also propose different roles of opioid, non-opioid DYN and orexin peptides on food choice and intake when palatable and non-palatable food options are available., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

We aimed to estimate the population fraction of poor early child health and developmental outcomes attributable to maternal depressive symptoms (DS) contrasting it between low- and middle/high-income households. We used a nationally representative probabilistic sample of 4240 children younger than 5 years old and their mothers, derived from the Mexican National Health and Nutrition Survey Data (ENSANUT 2012). Complex survey design, sampling, and analytic weights were taken into account in analyses. DS was measured by CESD-7. Child outcomes were as follows: breastfeeding, attending well-child check-ups, respiratory disease, diarrhea and general health problems, immunization, accidents, growth, obesity, and food insecurity. Prevalence of DS among mothers was 21.36%. In low-SES households, DS was associated with higher risk of never being breastfed (RR = 1.77; p < .05), health problems (RR = 1.37; p < .05), acute respiratory disease (RR = 1.51; p < .05), accidents requiring child hospitalization (RR = 2.16; p < .01), and moderate or severe food insecurity (RR = 1.58; p < .001). In medium- or high-SES households, DS was associated with higher risk of never attending a developmental check-up (RR = 2.14; p < .05) and moderate or severe food insecurity (RR = 1.75; p < .01). Population risks attributable to DS ranged from 2.30 to 17.45%. Prevention of DS could lead to reduction of problematic early childhood outcomes in both low and medium/high SES.

Objective:: To describe the methodology and the implementation survey results from National Survey of Children and Women Mexico's (ENIM 2015)., Materials and Methods:: The ENIM 2015 is a probability survey with multistage, stratified and cluster sample, with regional, rural and urban strata, and indigenous population representation.We applied questionnaires to get information from the household, women aged 15 to 49 years, children under five years and children and adolescents aged 5-17 years., Results:: The response rate for households and women was 94%, obtaining information from 10 760 households and 12 110 women; while for children and adolescents and children under five years was 98%, 11 607 and 8 066, respectively., Conclusion:: The ENIM 2015 probabilistic design allows generate indicators that can be stratified into five regions, rural and urban strata and from indigenous population, as well as a baseline for 15 indicators of the ODS.

Both iron overload and iron deficiency have been associated with cardiomyopathy and heart failure, but cardiac iron utilization is incompletely understood. We hypothesized that the transferrin receptor (Tfr1) might play a role in cardiac iron uptake and used gene targeting to examine the role of Tfr1 in vivo. Surprisingly, we found that decreased iron, due to inactivation of Tfr1, was associated with severe cardiac consequences. Mice lacking Tfr1 in the heart died in the second week of life and had cardiomegaly, poor cardiac function, failure of mitochondrial respiration, and ineffective mitophagy. The phenotype could only be rescued by aggressive iron therapy, but it was ameliorated by administration of nicotinamide riboside, an NAD precursor. Our findings underscore the importance of both Tfr1 and iron in the heart, and may inform therapy for patients with heart failure., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Divalent metal-ion transporter-1 (DMT1) is a widely expressed iron-preferring membrane-transport protein that serves a critical role in erythroid iron utilization. We have investigated its role in intestinal metal absorption by studying a mouse model lacking intestinal DMT1 (i.e., DMT1(int/int)). DMT1(int/int) mice exhibited a profound hypochromic-microcytic anemia, splenomegaly, and cardiomegaly. That the anemia was due to iron deficiency was demonstrated by the following observations in DMT1(int/int) mice: 1) blood iron and tissue nonheme-iron stores were depleted; 2) mRNA expression of liver hepcidin (Hamp1) was depressed; and 3) intraperitoneal iron injection corrected the anemia, and reversed the changes in blood iron, nonheme-iron stores, and hepcidin expression levels. We observed decreased total iron content in multiple tissues from DMT1(int/int) mice compared with DMT1(+/+) mice but no meaningful change in copper, manganese, or zinc. DMT1(int/int) mice absorbed (64)Cu and (54)Mn from an intragastric dose to the same extent as did DMT1(+/+) mice but the absorption of (59)Fe was virtually abolished in DMT1(int/int) mice. This study reveals a critical function for DMT1 in intestinal nonheme-iron absorption for normal growth and development. Further, this work demonstrates that intestinal DMT1 is not required for the intestinal transport of copper, manganese, or zinc., (Copyright © 2015 the American Physiological Society.)

Transferrin receptor (Tfr1) is ubiquitously expressed, but its roles in non-hematopoietic cells are incompletely understood. We used a tissue-specific conditional knockout strategy to ask whether skeletal muscle required Tfr1 for iron uptake. We found that iron assimilation via Tfr1 was critical for skeletal muscle metabolism, and that iron deficiency in muscle led to dramatic changes, not only in muscle, but also in adipose tissue and liver. Inactivation of Tfr1 incapacitated normal energy production in muscle, leading to growth arrest and a muted attempt to switch to fatty acid β oxidation, using up fat stores. Starvation signals stimulated gluconeogenesis in the liver, but amino acid substrates became limiting and hypoglycemia ensued. Surprisingly, the liver was also iron deficient, and production of the iron regulatory hormone hepcidin was depressed. Our observations reveal a complex interaction between iron homeostasis and metabolism that has implications for metabolic and iron disorders.

Background and Aims: Anastomotic leaks (AL) continue to be a devastating complication after colorectal surgery. The purpose of this experimental study was to confirm if Pebisut® applied to intestinal suture lines increases tensile strength in the critical days of healing and to evaluate its anti-inflammatory properties., Methods: Bursting pressures and histological evaluation of suture lines in dogs were performed, comparing the burst strength with collagen or fatty tissue patches with/without Pebisut® (patent granted in the USPTO 8,252.333, 26.01.2006, in the European Union 2,062,602, 01.12.2010, in Canada 2,661,686, 21.08.2007 and in Mexico P.C.T./MX/a/2009/001737)., Results: Pebisut® significantly increases burst strength in suture lines in long-term procedures with both collagen and fat pad patches. The adhesive penetrates rapidly into the suture lines, sealing them and progressing towards the intestinal lumen, disappearing in 14-20 days. It was well tolerated without any evidence of "foreign body" reaction., Conclusions: Application of the biodegradable adhesive Pebisut® is easy, well tolerated by mammalian tissues and consistently increases the burst strength of suture lines. Therefore, it may provide more tensile strength in anastomosis and help protect AL, one of the most serious complications in gastrointestinal surgery. If this experimental finding could be translated to clinical surgery, the protection of colorectal anastomosis could be beneficial to patients. Additionally, this could also have a positive impact on the economic expenditures of healthcare systems and patients., (Copyright © 2014 IMSS. Published by Elsevier Inc. All rights reserved.)

Transition metals are frequently used as cofactors for enzymes and oxygen-carrying proteins that take advantage of their propensity to gain and lose single electrons. Metals are particularly important in mitochondria, where they play essential roles in the production of ATP and detoxification of reactive oxygen species. At the same time, transition metals (particularly Fe and Cu) can promote the formation of harmful radicals, necessitating meticulous control of metal concentration and subcellular compartmentalization. We summarize our current understanding of Fe and Cu in mammalian mitochondrial biology and discuss human diseases associated with aberrations in mitochondrial metal homeostasis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Prolonged deficits in neural input activate pathological muscle remodeling, leading to atrophy. In denervated muscle, activation of the atrophy program requires HDAC4, a potent repressor of the master muscle transcription factor MEF2. However, the signaling mechanism that connects HDAC4, a protein deacetylase, to the atrophy machinery remains unknown. Here, we identify the AP1 transcription factor as a critical target of HDAC4 in neurogenic muscle atrophy. In denervated muscle, HDAC4 activates AP1-dependent transcription, whereas AP1 inactivation recapitulates HDAC4 deficiency and blunts the muscle atrophy program. We show that HDAC4 activates AP1 independently of its canonical transcriptional repressor activity. Surprisingly, HDAC4 stimulates AP1 activity by activating the MAP kinase cascade. We present evidence that HDAC4 binds and promotes the deacetylation and activation of a key MAP3 kinase, MEKK2. Our findings establish an HDAC4-MAPK-AP1 signaling axis essential for neurogenic muscle atrophy and uncover a direct crosstalk between acetylation- and phosphorylation-dependent signaling cascades., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background and Aims: Anastomotic leaks continue to be a devastating complication for patients and surgeons worldwide. The few surgical adhesives available to date have not achieved the desired clinical results. The purpose of this experimental study was to determine if Pebisut® applied to intestinal suture lines provides increased resistance and protection during the critical days of healing., Methods: Intestinal lesions were caused in rats and dogs and a new biodegradable adhesive (Pebisut®) (patent granted in the European Union 07808494.4-1219, 01.12.2010, in Mexico P.C.T./MX/a/2009/001737, 16.02.2009, pending in the U.S.P.T.O. 60/762,136, 26.01.2006) was applied to compare the resistance of suture lines using bursting pressures and histologically., Results: Under acute and chronic conditions, Pebisut® strengthened and made the suture lines more resistant, while histologically penetrating and sealing them. The adhesive disappears within 2-3 weeks and is well tolerated by the intestinal tissues., Conclusions: This biodegradable adhesive provides greater resistance, temporarily protects suture lines and may prevent anastomotic leaks., (Copyright © 2011 IMSS. Published by Elsevier Inc. All rights reserved.)

Histone deacetylase 4 (HDAC4) binds and inhibits activation of the critical muscle transcription factor myocyte enhancer factor-2 (MEF2). However, the physiological significance of the HDAC4-MEF2 complex in skeletal muscle has not been established. Here we show that in skeletal muscle, HDAC4 is a critical modulator of MEF2-dependent structural and contractile gene expression in response to neural activity. We present evidence that loss of neural input leads to concomitant nuclear accumulation of HDAC4 and transcriptional reduction of MEF2-regulated gene expression. Cell-based assays show that HDAC4 represses structural gene expression via direct binding to AT-rich MEF2 response elements. Notably, using both surgical denervation and the neuromuscular disease amyotrophic lateral sclerosis (ALS) model, we found that elevated levels of HDAC4 are required for efficient repression of MEF2-dependent structural gene expression, indicating a link between the pathological induction of HDAC4 and subsequent MEF2 target gene suppression. Supporting this supposition, we show that ectopic expression of HDAC4 in muscle fibers is sufficient to induce muscle damage in mice. Our study identifies HDAC4 as an activity-dependent regulator of MEF2 function and suggests that activation of HDAC4 in response to chronically reduced neural activity suppresses MEF2-dependent gene expression and contributes to progressive muscle dysfunction observed in neuromuscular diseases.

The composition of skeletal muscle, in terms of the relative number of slow- and fast-twitch fibers, is tightly regulated to enable an organism to respond and adapt to changing physical demands. The phosphatase calcineurin and its downstream targets, transcription factors of the nuclear factor of activated T cells (NFAT) family, play a critical role in this process by promoting the formation of slow-twitch, oxidative fibers. Calcineurin binds to calsarcins, a family of striated muscle-specific proteins of the sarcomeric Z-disc. We show here that mice deficient in calsarcin-2, which is expressed exclusively by fast-twitch muscle and encoded by the myozenin 1 (Myoz1) gene, have substantially reduced body weight and fast-twitch muscle mass in the absence of an overt myopathic phenotype. Additionally, Myoz1 KO mice displayed markedly improved performance and enhanced running distances in exercise studies. Analysis of fiber type composition of calsarcin-2-deficient skeletal muscles showed a switch toward slow-twitch, oxidative fibers. Reporter assays in cultured myoblasts indicated an inhibitory role for calsarcin-2 on calcineurin, and Myoz1 KO mice exhibited both an excess of NFAT activity and an increase in expression of regulator of calcineurin 1-4 (RCAN1-4), indicating enhanced calcineurin signaling in vivo. Taken together, these results suggest that calsarcin-2 modulates exercise performance in vivo through regulation of calcineurin/NFAT activity and subsequent alteration of the fiber type composition of skeletal muscle.

Histone deacetylase inhibitors (HDACI) are promising antitumor agents. Although transcriptional deregulation is thought to be the main mechanism underlying their therapeutic effects, the exact mechanism and targets by which HDACIs achieve their antitumor effects remain poorly understood. It is not known whether any of the HDAC members support robust tumor growth. In this report, we show that HDAC6, a cytoplasmic-localized and cytoskeleton-associated deacetylase, is required for efficient oncogenic transformation and tumor formation. We found that HDAC6 expression is induced upon oncogenic Ras transformation. Fibroblasts deficient in HDAC6 are more resistant to both oncogenic Ras and ErbB2-dependent transformation, indicating a critical role for HDAC6 in oncogene-induced transformation. Supporting this hypothesis, inactivation of HDAC6 in several cancer cell lines reduces anchorage-independent growth and the ability to form tumors in mice. The loss of anchorage-independent growth is associated with increased anoikis and defects in AKT and extracellular signal-regulated kinase activation upon loss of adhesion. Lastly, HDAC6-null mice are more resistant to chemical carcinogen-induced skin tumors. Our results provide the first experimental evidence that a specific HDAC member is required for efficient oncogenic transformation and indicate that HDAC6 is an important component underlying the antitumor effects of HDACIs.

TBX3 is a transcription factor of the T-box gene family. Mutations in the TBX3 gene can cause hypoplastic or absent mammary glands. Previous studies have shown that TBX3 might be associated with breast cancer. Here, we show that TBX3 is overexpressed in malignant cells of primary breast cancer tissues by immunohistochemistry. TBX3 interacts with histone deacetylases (HDAC) 1, 2, 3, and 5. TBX3 interacts with HDAC1, 2, and 3 via two distinct binding sites. However, deletion of the repression domain (amino acids 566-624) of TBX3 completely abolishes its interaction with HDAC5. Endogenous TBX3 and HDACs interaction and colocalization are found in a breast cancer cell line by coimmunoprecipitation and immunofluorescence, respectively. The functional significance of the interaction between TBX3 and HDAC is also tested in a p14(ARF)-luciferase reporter system. Results indicate that TBX3 represses expression of p14(ARF) tumor suppressor and that a HDAC inhibitor is able to reverse the TBX3 repressive function in a dosage-dependent manner. This study suggests that TBX3 may function by recruiting HDACs to the T-box binding site in the promoter region. TBX3 repression to its targets is dependent on HDAC activity. TBX3 may serve as a biomarker for breast cancer and have significant applications in both breast cancer diagnosis and treatment.

Neural activity actively regulates muscle gene expression. This regulation is crucial for specifying muscle functionality and synaptic protein expression. How neural activity is relayed into nuclei and connected to the muscle transcriptional machinery, however, is not known. Here we identify the histone deacetylase HDAC4 as the critical linker connecting neural activity to muscle transcription. We found that HDAC4 is normally concentrated at the neuromuscular junction (NMJ), where nerve innervates muscle. Remarkably, reduced neural input by surgical denervation or neuromuscular diseases dissociates HDAC4 from the NMJ and dramatically induces its expression, leading to robust HDAC4 nuclear accumulation. We present evidence that nuclear accumulated HDAC4 is responsible for the coordinated induction of synaptic genes upon denervation. Inactivation of HDAC4 prevents denervation-induced synaptic acetyl-choline receptor (nAChR) and MUSK transcription whereas forced expression of HDAC4 mimics denervation and activates ectopic nAChR transcription throughout myofibers. We determined that HDAC4 executes activity-dependent transcription by regulating the Dach2-myogenin transcriptional cascade where inhibition of the repressor Dach2 by HDAC4 permits the induction of the transcription factor myogenin, which in turn activates synaptic gene expression. Our findings establish HDAC4 as a neural activity-regulated deacetylase and a key signaling component that relays neural activity to the muscle transcriptional machinery.

Patients with Down syndrome have characteristic heart valve lesions resulting from endocardial cushion defects. The Down syndrome critical region 1 (DSCR1) gene, identified at the conserved trisomic 21 region in those patients, encodes a calcineurin inhibitor that inactivates nuclear factor of activated T cells (NFATc) activity. Here, we identify a regulatory sequence in the promoter region of human DSCR1 that dictates specific expression of a reporter gene in the endocardium, defined by the temporal and spatial expression of Nfatc1 during heart valve development. Activation of this evolutionally conserved DSCR1 regulatory sequence requires calcineurin and NFATc1 signaling in the endocardium. NFATc1 proteins bind to the regulatory sequence and trigger its enhancer activity. NFATc1 is sufficient to induce the expression of Dscr1 in cells that normally have undetectable or minimal NFATc1 or DSCR1. Pharmacologic inhibition of calcineurin or genetic Nfatc1 null mutation in mice abolishes the endocardial activity of this DSCR1 enhancer. Furthermore, in mice lacking endocardial NFATc1, the endogenous Dscr1 expression is specifically inhibited in the endocardium but not in the myocardium. Thus, our studies indicate that the DSCR1 gene is a direct transcriptional target of NFATc1 proteins within the endocardium during a critical window of heart valve formation.

In addition to regulating cell motility, contractility, and cytokinesis, the actin cytoskeleton plays a critical role in the regulation of transcription and gene expression. We have previously identified a novel muscle-specific actin-binding protein, STARS (striated muscle activator of Rho signaling), which directly binds actin and stimulates serum-response factor (SRF)-dependent transcription. To further dissect the STARS/SRF pathway, we performed a yeast two-hybrid screen of a skeletal muscle cDNA library using STARS as bait, and we identified two novel members of the ABLIM protein family, ABLIM-2 and -3, as STARS-interacting proteins. ABLIM-1, which is expressed in retina, brain, and muscle tissue, has been postulated to function as a tumor suppressor. ABLIM-2 and -3 display distinct tissue-specific expression patterns with the highest expression levels in muscle and neuronal tissue. Moreover, these novel ABLIM proteins strongly bind F-actin, are localized to actin stress fibers, and synergistically enhance STARS-dependent activation of SRF. Conversely, knockdown of endogenous ABLIM expression utilizing small interfering RNA significantly blunted SRF-dependent transcription in C2C12 skeletal muscle cells. These findings suggest that the members of the novel ABLIM protein family may serve as a scaffold for signaling modules of the actin cytoskeleton and thereby modulate transcription.

Myocardin and the myocardin-related transcription factors (MRTFs) MRTF-A and MRTF-B are coactivators for serum response factor (SRF), which regulates genes involved in cell proliferation, migration, cytoskeletal dynamics, and myogenesis. MRTF-A has been shown to translocate to the nucleus and activate SRF in response to Rho signaling and actin polymerization. Previously, we described a muscle-specific actin-binding protein named striated muscle activator of Rho signaling (STARS) that also activates SRF through a Rho-dependent mechanism. Here we show that STARS activates SRF by inducing the nuclear translocation of MRTFs. The STARS-dependent nuclear import of MRTFs requires RhoA and actin polymerization, and the actin-binding domain of STARS is necessary and sufficient for this activity. A knockdown of endogenous STARS expression by using small interfering RNA significantly reduced SRF activity in differentiated C2C12 skeletal muscle cells and cardiac myocytes. The ability of STARS to promote the nuclear localization of MRTFs and SRF-mediated transcription provides a potential muscle-specific mechanism for linking changes in actin dynamics and sarcomere structure with striated muscle gene expression.

Signaling by the calcium-dependent phosphatase calcineurin profoundly influences the growth and gene expression of cardiac and skeletal muscle. Calcineurin binds to calsarcins, a family of muscle-specific proteins of the sarcomeric Z-disc, a focal point in the pathogenesis of human cardiomyopathies. We show that calsarcin-1 negatively modulates the functions of calcineurin, such that calcineurin signaling was enhanced in striated muscles of mice that do not express calsarcin-1. As a consequence of inappropriate calcineurin activation, mice with a null mutation in calsarcin-1 showed an excess of slow skeletal muscle fibers. The absence of calsarcin-1 also activated a hypertrophic gene program, despite the absence of hypertrophy, and enhanced the cardiac growth response to pressure overload. In contrast, cardiac adaptation to other hypertrophic stimuli, such as chronic catecholamine stimulation or exercise, was not affected. These findings show important roles for calsarcins as modulators of calcineurin signaling and the transmission of a specific subset of stress signals leading to cardiac remodeling in vivo.

Graft rejection after T cell-depleted HLA-genotypically identical bone marrow transplantation (BMT) is probably mediated by mH antigen-specific cytotoxic T lymphocytes (CTL). We have analyzed peripheral blood mononuclear cells (PBMC) from a female bone marrow graft recipient, collected during graft rejection after a sex mismatched HLA-identical BMT. A CTL line was generated by stimulating recipient PBMC collected during graft rejection with donor PBMC and donor EBV-transformed lymphoblastoid cell lines. From this CTL line a large number of clones of different specificity and phenotype was established by limiting dilution. These clones exhibited several mH antigen specificities, restricted by HLA-B7, -B27 or -DR2 as shown by differential recognition of family members and unrelated individuals sharing potential restriction elements. The CD3+CD4+ and CD3+CD8+ bulk culture was cloned, resulting in 50 HLA-B7 restricted CD3+CD4-CD8+CTL clones, three HLA-B27 restricted CD3+CD4-CD8+CTL clones, one HLA-DR2 restricted CD3+CD4+CD8-CTL clone and two additional HLA class II restricted CD3+CD4+CD8-CTL clones with a different specificity. One representative clone of each specificity was selected for further analysis. The CTL line and the HLA-B7 restricted CD8+CTL clone, but not the HLA class II restricted CD4+ CTL clone, inhibited the growth of donor hematopoietic progenitor cells (HPC). In conclusion, these results show that graft rejection after HLA-identical BMT may be mediated by multiple CTL clones that specifically recognize one mH antigen peptide presented by different HLA molecules or different mH antigens expressed on donor cells and that CTL, but not CD4+ CTL inhibited donor HPC growth.

Endogenously produced and exogenously administered granulocyte colony-stimulating factor (G-CSF) has correlated with myeloid engraftment in a number of hematopoietic progenitor cell transplantation settings. Given the increased susceptibility of T cell-depleted (TCD) bone marrow transplants (BMT) to graft failure, a cohort of 36 (21 male and 15 female) recipients of TCD BMT was evaluated prospectively during the first month post-transplant for circulating serum G-CSF levels, to examine the correlation between myeloid engraftment and G-CSF levels. All recipients of TCD BM had measurable G-CSF levels, with a median peak level of 1750 pg/ml (range 540-26,250 pg/ml) occurring at a median of 5 days (range 1-18 days) after BM infusion. There was no association between G-CSF kinetics within 1 month post-transplant and the development of primary non-engraftment or secondary graft failure. One patient with primary non-engraftment and 6 patients with secondary graft failure exhibited median G-CSF peak levels of 1600 pg/ml and 1850 pg/ml (range 600-16,250 pg/ml) occurring 5 and 5.5 days (range 4-7 days) after BM infusion, respectively. Additionally, the patient with primary non-engraftment demonstrated a high G-CSF level in response to a low absolute neutrophil count (ANC). An inverse relationship between serial G-CSF levels and concomitant ANC was documented (log G-CSF = 6.19-0.009 ANC, P < 0.001). Higher peak G-CSF levels were associated with older recipient age (P = 0.01) and lower BM cell dose (P = 0.02), while administration of anti-thymocyte globulin post-transplant did not alter G-CSF levels.

Engraftment of marrow following autologous or allogeneic bone marrow transplantation (BMT) may be influenced by quantity and function of stem cells. T lymphocytes, supporting microenvironmental cells, and hematopoietic growth factors (HGF). To elucidate the physiologic role of interleukin-3 (IL-3) in the engraftment process, serum IL-3 levels were measured in over 400 samples from 77 transplant recipients before and for up to 3 weeks following transplantation using a novel enzyme-linked immunoabsorbent assay (ELISA) with a sensitivity of > or = 78 pg/mL. Thirty-seven patients received two to three log T-cell-depleted allografts. In the remaining 40 patients (18 autologous marrow, 12 allogeneic marrow, and 10 autologous peripheral blood [PB] stem cell), T cells were not depleted (non-TCD) from the grafts. A burst of IL-3 (peak levels, 1,500 to 6,000 pg/mL) was detected in the immediate posttransplant period between day 0 and day 14 in all non-TCD recipients and in 21 of 37 (57%) of TCD recipients. A strong inverse relationship between IL-3 levels and absolute neutrophil count (ANC) was observed in both non-TCD recipients (r = -.796) and in TCD recipients (r = -.897). However, both peak IL-3 levels and mean IL-3 levels from day 0 through 14 were significantly lower in TCD recipients compared with either autologous or unmodified allogeneic marrow recipients (P < .01). The lowest peak or mean day 0 through 14 IL-3 levels were observed in matched related recipients undergoing the most aggressive (2.5 to 3.0 log) T-cell-depleted BMT. Autografted patients receiving blood stem cell transplants alone or posttransplant granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony stimulating factor (GM-CSF) also had significantly lower peak IL-3 levels (P < .01). In patients receiving TCD grafts, administration of antithymocyte globulin (ATG) posttransplant significantly increased peak IL-3 levels compared with patients not treated with ATG (P < .04). This study shows that endogenous release of IL-3 is strongly associated with myeloid engraftment and inversely related to ANC. Removal of T lymphocytes from donor marrow or acceleration of engraftment by use of stem cells or growth factors appears to blunt the endogenous release of IL-3 whereas use of ATG posttransplant increases IL-3 release.

Since the water reserve of the interstitium plays a major role in circulatory homeostasis, its reduction by dehydration may produce severe changes in the organism's response to hemorrhage but this has not been measured experimentally. Twelve immature pigs (22 +/= 2 Kg) were divided into two groups of six each. Control animals had free access to food and water prior to bleeding. Dehydrated animals had water withheld for 48 hours preceding the bleeding. All animals were bled 30 per cent of their calculated blood volumes while awake. No resuscitation was performed. No mortality was observed in the control group of animals, while four of the six dehydrated animals died (66%). All deaths occurred between one and four hours posthemorrhage. Plasma refill reached 33 per cent by 0.5 hours in the control group compared to only 17 per cent by 0.5 hours in the dehydrated group (p less than or equal to .05). Refill in the control group reached 50 per cent by three hours, whereas dehydrated animals surviving to three hours demonstrated no further refill (p less than or equal to .05). BUN, calcium, sodium, and osmolality were consistently higher in dehydrated than control animals (p less than or equal to .05). It is concluded that a reduction in the interstitial water reserve significantly impairs ability to recover from hemorrhage.