Use of AgomiR and AntagomiR technologies to alter satellite cell proliferation in vitro, miRNA expression, and muscle fiber hypertrophy in intrauterine growth-restricted lambs.

Introduction: microRNAs (miRNAs) are small non-coding RNAs that work at the posttranscriptional level to repress gene expression. Several miRNAs are preferentially expressed in skeletal muscle and participate in myogenesis. This research was conducted to alter endogenous miRNA expression in skeletal muscle to promote muscle hypertrophy. Methods: Two experiments were conducted using mimic/agomiR or antagomir technologies to alter miRNA expression and examine changes in myoblast proliferation in vitro (experiment 1) and muscle hypertrophy in vivo (experiment 2). In vitro experiments found that antagomiR-22-3p and mimic-127 increased myoblast proliferation compared to other miRNA treatments or controls. These miRNA treatments, antagomiR-22-3p (ANT22) and agomiR-127 (AGO127), were then used for intramuscular injections in longissimus muscle. Results and discussion: The use of antagomiR or mimic/agomiR treatments down-regulated or up-regulated, respectively, miRNA expression for that miRNA of interest. Expression of predicted target KIF3B mRNA for miR-127 was up-regulated and ACVR2a mRNA was up-regulated for miR-22-3p. ANT22 injection also up-regulated the major regulator of protein synthesis (mTOR). Proteomic analyses identified 11 proteins for AGO127 and 9 proteins for ANT22 that were differentially expressed. Muscle fiber type and cross-sectional area were altered for ANT22 treatments to transition fibers to a more oxidative state. The use of agomiR and antagomir technologies allows us to alter miRNA expression in vitro and in vivo to enhance myoblast proliferation and alter muscle fiber hypertrophy in IUGR lambs during early postnatal growth.

miRNA transcriptome and myofiber characteristics of lamb skeletal muscle during hypertrophic growth1.

Postnatal muscle growth is achieved through hypertrophy of the muscle fibers and is impacted by the activity of satellite cells, the quiescent muscle stem cell. Several miRNAs are preferentially expressed in skeletal muscle and could provide a mechanism for increasing muscle hypertrophy through satellite cell proliferation and/or differentiation. The objectives of this study were to: 1) Characterize the miRNA transcriptome of the longissimus thoracis et lumborum muscle at several developmental timepoints [gestational d 85 (PN1), 110 (PN2), 133 (PN3), postnatal d 42 (PW1), 65 (PW2), 243 (MAT)] during muscle hypertrophy in lambs, and 2) examine miR-29a, identified in sequencing to be differentially regulated across development, loss of function on satellite cell proliferation and differentiation. Muscle fiber characteristics showed drastic increases (p < 0.0001) in fiber size and alterations in muscle fiber type occur during pre and postnatal development. miRNA sequencing comparisons were performed in developmental order (PN1 vs. PN2, PN2 vs. PN3, PN3 vs. PW1, PW1 vs. PW2, PW2 vs. MAT). There were 184 differentially expressed (P adj < 0.05) miRNA, 142 unique miRNA, from all 5 comparisons made. The transitional stage (PN3 vs. PW1) had the largest number (115) of differentially expressed miRNA. Inhibition of miR-29a in satellite cell culture increased (p < 0.05) cell proliferation and differentiation capacity. Characterization of the miRNA transcriptome provides valuable insights into the miRNA involved in muscle fiber hypertrophy and the potential importance of the transitional period.

Mycotoxin ingestion during late gestation alters placentome structure, cotyledon transcriptome, and fetal development in pregnant sheep.

Ergot alkaloids, a class of mycotoxins, induce vasoconstriction when consumed by animals and humans. Pregnant ewes (n = 16; 81.2 kg ± 7.7) were assigned fed endophyte-infected tall fescue seed (E+; 4.14 µg ergovaline + ergovalinine/g seed) or a control diet (CON; 0 µg ergovaline + ergovalinine) for increasing duration during late gestation (from gd86 to gd110 or gd133) to examine changes in placentome morphology and mRNA transcriptome, and fetal development. Exposure to E+ fescue reduced serum prolactin concentrations at gd110 and gd133 demonstrating treatment efficacy. For control ewes, cotyledon and total placentome weights decreased with advancing gestation due to remodeling of placental tissues; however, cotyledon and placentome weight did not change with advancing gestation in E+ fed ewes. Fetal brain sparing was evident in E+ exposed fetuses at gd110 and gd133 compared to CON, which demonstrates asymmetrical growth and intrauterine growth restriction. Mycotoxin exposure (E+) resulted in differential expression of 22 genes in the cotyledon tissue at gd110 but only one gene at gd133 compared to CON. These results suggest that the response to mycotoxin exposure in the pregnant sheep model has an immediate impact on placental remodeling and fetal development that persists throughout the duration of the exposure period.

Impact of fetal exposure to mycotoxins on longissimus muscle fiber hypertrophy and miRNA profile.

BACKGROUND: Longissimus muscle samples were collected from lambs exposed in utero to mycotoxins [E-, endophyte-free tall fescue seed without ergot alkaloids (negative control) or E + , endophyte-infected tall fescue seed containing ergot alkaloids] during mid-gestation (MID; E + /E-) or late-gestation (LATE; E-/E +) harvested at two developmental stages (FETAL, gestational d133) or (MAT, near maturity, 250 d of age; n = 3/treatment/developmental stage). Muscle samples were examined to determine the impact of in utero mycotoxin exposure on skeletal muscle fiber hypertrophy and the miRNA profile at FETAL and MAT. RESULTS: Longissimus weight was greater (P < 0.05) in E + /E- lambs compared to E-/E + lambs at MAT; however, FETAL longissimus weight did not differ (P > 0.10) between fescue treatments. Type I fiber cross sectional area was larger (P < 0.10) for E + /E- than E-/E + at MAT but did not differ (P > 0.10) between fescue treatments at FETAL. Type II fiber area was larger (P < 0.05) at MAT in E + /E- compared to E-/E + but did not differ (P < 0.05) between fescue treatments at FETAL. Cross-sectional Type I and Type II longissimus muscle fiber area increased (P < 0.05) from FETAL to MAT by 6.86-fold and 10.83-fold, respectively. The ratio of Type II:Type I muscle fibers was lower (P = 0.04) at MAT compared to FETAL. There were 120 miRNA differentially expressed (q < 0.05) between FETAL and MAT. Maternal fescue treatment did not alter (q > 0.05) expression of miRNAs in the longissimus muscle. miR-133, -29a, -22-3p, and -410-3p were identified as highly significant with a log2 fold change > 4. In vitro satellite cell cultures showed that selected miRNAs (miR-22-3p, 29a, 27a, and 133a) are differentially regulated during proliferation and differentiation indicating a role of miRNA in muscle hypertrophy. CONCLUSIONS: Exposure to mycotoxins did not alter fiber type but had long-term impacts on postnatal muscle hypertrophy and cross-sectional area. The miRNA profile of the longissimus was not altered by Maternal mycotoxin exposure at FETAL or MAT. Developmental age altered the miRNA transcriptome and mRNA expression of known genes related to muscle growth. These results indicate that Maternal exposure to E + fescue seed during LATE gestation can alter postnatal muscle hypertrophy in sheep; however, these changes are not regulated by the miRNA transcriptome of the longissimus muscle.

Aging-related inflammation in osteoarthritis.

It is well accepted that aging is an important contributing factor to the development of osteoarthritis (OA). The mechanisms responsible appear to be multifactorial and may include an age-related pro-inflammatory state that has been termed "inflamm-aging." Age-related inflammation can be both systemic and local. Systemic inflammation can be promoted by aging changes in adipose tissue that result in increased production of cytokines such as interleukin (IL)-6 and tumor necrosis factor-α (TNFα). Numerous studies have shown an age-related increase in blood levels of IL-6 that has been associated with decreased physical function and frailty. Importantly, higher levels of IL-6 have been associated with an increased risk of knee OA progression. However, knockout of IL-6 in male mice resulted in worse age-related OA rather than less OA. Joint tissue cells, including chondrocytes and meniscal cells, as well as the neighboring infrapatellar fat in the knee joint, can be a local source of inflammatory mediators that increase with age and contribute to OA. An increased production of pro-inflammatory mediators that include cytokines and chemokines, as well as matrix-degrading enzymes important in joint tissue destruction, can be the result of cell senescence and the development of the senescence-associated secretory phenotype (SASP). Further studies are needed to better understand the basis for inflamm-aging and its role in OA with the hope that this work will lead to new interventions targeting inflammation to reduce not only joint tissue destruction but also pain and disability in older adults with OA.

Function of the chondrocyte PI-3 kinase-Akt signaling pathway is stimulus dependent.

OBJECTIVE: The PI-3 kinase-Akt pathway plays a role in cartilage anabolic as well as catabolic processes in response to activation by insulin-like growth factor-1 (IGF-1) and the pro-inflammatory cytokines interleukin-1ß (IL-1ß) and oncostatin M (OSM). The goal of this study was to determine how PI-3 kinase-Akt signaling regulates these seemingly opposing functions. DESIGN: Monolayer cultures of primary human articular chondrocytes were treated with IGF-1, IL-1ß, OSM, or the combination of IL-1ß and OSM in time course experiments. Activation of signaling proteins and MMP production were measured by immunoblotting. Cells were pre-treated with chemical inhibitors to block mitogen activated protein (MAP) kinases, PI-3 kinase, or JAK/STAT pathway activation. Constitutively active Akt1 and Akt3 were expressed to study stimulus-independent activation of Akt. RESULTS: IGF-1, OSM, and the combination of IL-1ß and OSM but not IL-1ß alone, stimulated phosphorylation of Akt which was sustained longer with IGF-1. IL-1ß plus OSM, but not IGF-1, increased chondrocyte MMP-13 production which was inhibited with either a general PI-3 kinase inhibitor or specific inhibition of the PI-3 kinase-γ isoform. Akt1 or Akt3 activity alone was not sufficient to increase production of MMP-13. IL-1ß/OSM induced MMP-13 production required activation of the MAP kinases, JNK and p38, as well as the JAK-STAT pathway which were activated by IL-1ß plus OSM but not by IGF-1. CONCLUSIONS: The chondrocyte integrates signals from the PI-3 kinase-Akt pathway with signals from MAP kinases and the JAK-STAT pathway to allow for a differential response to a pro-anabolic (IGF-1) and a pro-catabolic (IL-1ß plus OSM) stimulus.

Study of the effectiveness of the US safety standard for child resistant cigarette lighters.

OBJECTIVE: The purpose of this research is to evaluate the effectiveness of the US Consumer Product Safety Commission's (CPSC) Safety Standard for Cigarette Lighters, which requires that disposable cigarette lighters be resistant to operation by children younger than age 5. METHODS: Fire data on children playing with lighters were solicited from selected US fire departments for incidents occurring from 1997-99, to identify the proportion of such fires caused by children younger than age 5 playing with cigarette lighters. These data were compared with similar data from 1985-87. An odds ratio was used to determine if there was a significant decrease in cigarette lighter fires caused by children younger than age 5 compared to children ages 5 and older. To estimate fires that would have occurred without the standard, the odds ratio, adjusted for population, was applied to 1998 national estimates of fires occurring. National estimates of 1998 fire losses were based on data from the National Fire Incident Reporting System and the National Fire Protection Association to which the 1997-99 age and lighter type distributions were applied. The difference between the fire losses that would have occurred and those that did occur represented fire losses prevented. RESULTS: In the post-standard study, 48% of the cigarette lighter fires were started by children younger than age 5, compared with 71% in the pre-standard study. The odds ratio of 0.42 was statistically significant (p<0.01). This represented a 58% reduction in fires caused by the younger age group compared to the older age group. When applied to national fire loss data, an estimated 3300 fires, 100 deaths, 660 injuries, and $52.5 million in property loss were prevented by the standard in 1998, totaling $566.8 million in 1998 societal savings. CONCLUSIONS: The CPSC standard requiring child resistant cigarette lighters has reduced fire deaths, injuries, and property loss caused by children playing with cigarette lighters and can be expected to prevent additional fire losses in subsequent years.

A pooled analysis of 10 case-control studies of melanoma and oral contraceptive use.

Data regarding the effects of oral contraceptive use on women's risk of melanoma have been difficult to resolve. We undertook a pooled analysis of all case-control studies of melanoma in women completed as of July 1994 for which electronic data were available on oral contraceptive use along with other melanoma risk factors such as hair colour, sun sensitivity, family history of melanoma and sun exposure. Using the original data from each investigation (a total of 2391 cases and 3199 controls), we combined the study-specific odds ratios and standard errors to obtain a pooled estimate that incorporates inter-study heterogeneity. Overall, we observed no excess risk associated with oral contraceptive use for 1 year or longer compared to never use or use for less than 1 year (pooled odds ratio (pOR)=0.86; 95% CI=0.74-1.01), and there was no evidence of heterogeneity between studies. We found no relation between melanoma incidence and duration of oral contraceptive use, age began, year of use, years since first use or last use, or specifically current oral contraceptive use. In aggregate, our findings do not suggest a major role of oral contraceptive use on women's risk of melanoma.

The effects of the introduction of medication on the psychoanalytic process: a case study.

The past fifteen years have seen a marked change in the attitude of psychoanalysts toward the use of medication in psychoanalysis, yet few detailed case studies have appeared in which the effects of the introduction of a medication on the psychoanalytic process have been studied. A plea for fluoxetine (Prozac) as a weight-loss aid, from a female patient whose analysis had progressed satisfactorily, set in motion a remarkable series of events. Her mood brightened, she rapidly lost weight, and she ultimately realized that she had the fantasy of being impregnated, all of which represented the reenactment of crucial childhood experiences. The most significant change in the psychoanalytic process was a newfound ability to experience critical thoughts and feelings, which previously had been prohibited by her harsh superego. At first both patient and analyst believed that the medication had "softened" her superego, but reexamination of the data revealed that most, if not all, of the changes could be attributed to the transference meaning of the collaboration rather than to the effects of the drug.

Arm complications after radial artery procurement for coronary bypass operation.

BACKGROUND: Radial arteries are being used frequently for coronary artery bypass conduits, and surgeons are appropriately concerned about the risks of ischemia, infection, and neurologic dysfunction of the "donor" arm. We present our record of donor site complications, with emphasis on the safety of radial usage in patients older than 65 years of age. METHODS: A total of 343 radial arteries were removed from 217 patients, aged 37 to 83 years. Forty-nine (23%) of the patients were women; 126 of the radial arteries were from patients aged 65 years or older. Four patients died. In the remaining 213 patients, 338 donor arms were examined for at least 6 weeks postoperatively for evidence of infection, ischemia, hematoma, seroma, or paresthesia. RESULTS: No donor arms developed symptoms of 'ischemia or motor dysfunction. Two of 338 (0.6%) arms developed hematomas requiring operative drainage. Relatively minor complications of stitch abscesses, skin dehiscence, superficial infection, and small hematomas or seromas affected another 14 arms (4.1%). Cutaneous paresthesias in the radial distribution of the lateral antebrachial cutaneous nerve or superficial branch of the radial nerve were noted postoperatively in 36 arms (10.7%). The risk of any arm complication occurring was 13% in the 65 and older group and 17% in those less than 65 years old (p = NS). CONCLUSIONS: The risk of a major arm complication from radial artery harvesting for coronary bypass is low, even in patients of advanced age.

Interdigital neuroma: intermuscular neuroma transposition compared with resection.

This prospective, randomized study compares the treatment of an interdigital neuroma (IDN) by the standard resection operation with a technique in which the IDN is transposed into the inter-muscular space between the adductor hallucis and the interossei muscles after division of the digital nerves distal to the IDN. The resection group contained 22 patients and 22 neuromas and the transposition group contained 22 patients and 23 neuromas. An interviewer, blinded as to the operative technique used, telephoned each patient preoperatively, and at 1 month, 3 months, 6 months, 12 months, and 36-48 months postoperatively. The interviewer recorded the patient's reported pain level on a numerical rating scale of 0 to 100. In the resection group the average pain level was slightly lower through the first 6 month period, but at the 12 month review the resection group had a slightly higher average pain level . At the 36-48 month survey the resection group again reported a greater average pain level and fewer asymptomatic patients. It was concluded that it is unnecessary to excise the IDN to obtain excellent relief of pain. It was also concluded that transposition of the IDN into an intermuscular position between the adductor hallucis and the interossei muscles produced significantly better long term results than did the standard resection operation.

Endoscopic evaluation of the esophagus in infants and children immediately following intraoperative use of transesophageal echocardiography.

OBJECTIVE: Intraoperative transesophageal echocardiography (TEE) has evolved as an essential technique for use during pediatric cardiac surgery; however, few studies have evaluated the safety of TEE in children. This series reports endoscopic examination of the esophagus following intraoperative TEE in pediatric patients. METHODS: Fifty children undergoing congenital heart surgery underwent flexible esophagoscopy that was performed after completion of their heart surgery and after the removal of the transesophageal echo probe. The patients' ages ranged from 4 days to 10 years old, and their weight ranged from 3.0 to 39.8 kg, with a mean weight of 12.6 kg. RESULTS: Thirty-two of 50 patients (64%) had abnormal results shown on esophageal examinations; this occurred more frequently in the subset of patients weighing < 9 kg. No long-term feeding or swallowing difficulties were noted in any of the 48 patients who survived. CONCLUSIONS: Intraoperative TEE in infants and children frequently caused mild mucosal injury. Care must be exercised in the insertion and manipulation of the probes.

Reductions in deaths in frontal crashes among right front passengers in vehicles equipped with passenger air bags.

CONTEXT: Virtually all new cars now are equipped with passenger air bags. Determining whether passenger air bags are saving lives is important, particularly because passenger air bags have caused some deaths among children and adults. OBJECTIVE: To assess the effectiveness of passenger air bags in reducing the risk of death in frontal crashes for right front passengers. DESIGN: Air bags are designed to protect occupants in frontal crashes. Using Fatality Analysis Reporting System data for calendar years 1992 through 1995, the relative frequency of right front passenger deaths in frontal vs nonfrontal fatal crashes was compared for cars with dual air bags and for cars with driver-only air bags. MAIN OUTCOME MEASURES: Odds of right front passengers dying in frontal compared with nonfrontal fatal crashes were computed for 1992 through 1995 model year cars with dual air bags and for cars with driver-only air bags. Percentage reductions in right front passenger deaths in dual air bag vehicles were estimated. RESULTS: Right front passenger fatalities were 18% lower than expected in frontal crashes of cars with dual air bags and 11% lower in all crashes. An estimated 73 fewer than expected right front passengers died in 1992 through 1995 model cars with dual air bags during 1992 through 1995. The risk of frontal crash death for right front passengers in cars with dual air bags was reduced 14% among those reported to be using belts and 23% among belt nonusers. Children younger than 10 years in cars with dual air bags had a 34% increased risk of dying in frontal crashes. CONCLUSIONS: Passenger air bags were associated with substantial reductions in fatalities among right front passengers in frontal crashes. However, more children are being killed than are being saved by air bags. Immediate countermeasures to reduce the dangers of air bags to children and adults are suggested.

Drinking drivers missed at sobriety checkpoints.

OBJECTIVE: Sobriety checkpoints are an effective deterrent to alcohol-impaired driving although a substantial proportion of drinking drivers who pass through checkpoints are missed. The present study was designed to determine the extent to which police officers correctly identify individuals with blood alcohol concentrations (BACs) at or above 0.05% at checkpoints, and if there are characteristics of drivers, vehicles or checkpoints that are associated with decreased chances of detection. METHOD: To determine which drivers are likely to be missed, drivers not detained by police for additional sobriety evaluation were interviewed and voluntary breath samples were provided at 156 sobriety checkpoints in North Carolina. RESULTS: More than 50% of the drivers with BACs in excess of 0.08% and almost 90% of drivers with BACs in excess of 0.05% were not detained by officers. For drivers with BACs of 0.05% or higher, women and those 35 or younger were more likely to be missed than were men and older drivers. Drivers without passengers were more likely to be missed than those with passengers. Drivers were also more likely to be missed during weekend checkpoints. Similar results were found for drivers with BACs at or above 0.08%. CONCLUSIONS: Alerting police officers to characteristics of drinking drivers more likely to be missed may improve detection rates. In addition, passive alcohol sensors could improve the effectiveness and efficiency of sobriety checkpoints in detecting drinking drivers.

Unilateral pulmonary agenesis, esophageal atresia, and distal tracheoesophageal fistula.

A small-for-gestational-age, premature infant with the combination of unilateral pulmonary agenesis, esophageal atresia, and distal tracheoesophageal fistula was treated successfully by early gastrostomy and delayed fistula division with esophagoesophagostomy. Only 2 other successfully treated cases have been reported previously. Both were full-term infants treated with early division of the tracheoesophageal fistula and esophagoesophagostomy. Gestational age, size, and associated medical problems need to be considered when planning operative therapy for these babies.

Resistance and modes of communication.

The recognition that there are three modes of thinking and communicating--iconic, enactive, and symbolic--clarifies and simplifies many issues in psychoanalytic theory and technique. Although this concept is applicable to all communication, it is particularly important in understanding and interpreting various types of resistance. The author uses a number of vignettes to illustrate the forms and functions of various types of nonverbal communication within the psychoanalytic process.

The changing strategies in operation for transposition of the great vessels.

Between July 3, 1985, and February 24, 1994, a total of 55 infants underwent arterial switch procedures for the repair of transposition of the great vessels. Thirty-five infants had an intact ventricular septum and 20 had ventricular septal defects. To date, there have been three late deaths, one in the group with an intact ventricular septum and two in the group with a ventricular septal defect. Early postoperative complications included atrial dysrhythmias, prolonged ventilation, inability to close the sternum, and tension on the coronary arteries. Follow-up echocardiographic data for 44 patients indicate that pulmonary artery gradients are a worrisome postoperative problem, especially in infants who have ventricular septal defects.

Avascular necrosis of the first metatarsal head: incidence in distal osteotomy combined with lateral soft tissue release.

The incidence of avascular necrosis of the metatarsal head following distal first metatarsal osteotomy combined with adductor tendon release has not been documented in a large series of patients. Of 82 consecutive procedures in 64 patients performed between 1986 and 1988, 42 patients (58 procedures) were available for clinical and radiographic examination. Average follow-up was 2.5 years (range 1.0-4.2 years). There were 35 L-shaped and 23 chevron osteotomies which were combined with a lateral soft tissue release that included adductor tenotomy. Preoperative hallux valgus angle averaged 25 degrees (range 15-40 degrees), and intermetatarsal angle averaged 12 degrees (range 5-24 degrees). Follow-up amount of correction averaged 13 degrees and 5 degrees, respectively. Eighty-four percent of patients were satisfied with their result. There was one case of avascular necrosis. The patient was asymptomatic at 4.2 years' follow-up, and the remaining patients included two with infections, one hallux varus, and no nonunions.