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Background: Many complex new procedures involve a learning curve, and patients treated by individuals who are new to a procedure may have more complications than those treated by a practitioner who has performed the intervention more frequently. Still, at some point on the learning curve, each individual must decide that he or she is qualified to perform a procedure, presumably on the basis of his or her level of confidence, background, education, and skill. To evaluate the interrelationship of these factors, we designed a study in which we assessed the performance of a simulated knee joint injection.Methods: Ninety-three practitioners attending a continuing medical education session on knee injection were randomized to receive skills instruction through the use of a printed manual, a video, or hands-on instruction; each performed one injection before and after instruction. The participants completed pre-instruction and post-instruction questionnaires gauging confidence and also provided self-assessments of their performances of injections before and after instruction. Self-assessments were compared with objective performance standards measured by custom-designed knee models with electronic sensors that detected correct needle placement.Results: Before instruction, the participants' confidence was significantly but inversely related to competent performance (r = -0.253, p = 0.02); that is, greater confidence correlated with poorer performance. Both men and physician-practitioners displayed higher pre-instruction confidence (p < 0.01), which was not correlated with better performance. After instruction, performance improved significantly in all three training groups (p < 0.001), with no significant differences in efficacy detected among the three groups (p = 0.99). After instruction, confidence correlated with objective competence in all groups (r = 0.24, p = 0.04); however, this correlation was weaker than the correlation between the participants' confidence and their self-assessment of performance (r = 0.72, p = 0.001).Conclusions: Even low-intensity forms of instruction improve individuals' confidence, competence, and self-assessment of their skill in performing the fairly straightforward psychomotor task of simulated knee injection. However, men and physicians disproportionately overestimated their skills both before and after training, a finding that worsened as confidence increased. The inverse relationship between confidence and competence that we observed before the educational intervention as well as the demographic differences that we noted should raise questions about how complex new procedures should be introduced and when self-trained practitioners should begin to perform them. [ABSTRACT FROM AUTHOR]

With a rising worldwide incidence of obesity, particularly in the young, bariatric surgery offers an effective method of meaningful and sustained weight loss. At present, most bariatric procedures are carried out in women and increasingly in younger age groups. In line with the fertility benefits associated with weight loss, pregnancy after bariatric surgery is now a very common scenario. Although there is limited evidence to support optimal care in this group, most women appear to have good pregnancy outcomes, with reduced rates of preeclampsia and gestational diabetes (GDM). However, rates of stillbirth and small-for gestational-age (SGA) babies are increased, suggesting that screening and supplementation of micronutrients is likely to be very important in this cohort. The risks and benefits bariatric surgery may pose to pregnancy outcomes, both maternal and fetal, are largely dependent upon the degree of weight loss, weight stability upon entering pregnancy, surgical complications and the time interval between bariatric surgery and pregnancy. Ideally, preconception care would be more widely available, helping to assess and address micronutrient deficiencies and support preparation for pregnancy., Competing Interests: Declaration of Competing Interests The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Claire L Meek reports a relationship with NIHR Leicester BRC that includes: funding grants. Claire L Meek reports a relationship with European Foundation for the Study of Diabetes that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

A universal response to cellular stress is the expression of transformation-related protein 53 (TRP53). This transcription factor reduces cell proliferation and/or survival and is classed as a tumour suppressor protein. Several stresses (including culture) cause increased TRP53 expression in blastocysts and their reduced long-term developmental potential. This study shows that culture from the zygote stage (but not the 2-cell stage) reduced the development of C57BL6 inbred (but not hybrid) strain mouse embryos. Reduced viability was TRP53 dependent, being partially reversed by a TRP53 inhibitor (Pifithrin-alpha). However, the presence of culture did not cause an increase in Trp53 mRNA levels (levels were reduced following culture, P < 0.001). Transformed mouse 3T3 cell double minute 2 (MDM2) causes the ubiquitination and degradation of TRP53. MDM2 activation is accompanied by phosphorylation of Ser-166, and this is commonly catalyzed by the phosphatidylinositol-3 kinase and RAC-alpha serine/threonine-protein kinase (AKT) signaling pathway. Paf is an autocrine embryotrophin that activates the phosphatidylinositol-3 kinase/AKT pathway. High levels of TRP53 expression occurred following the culture of zygotes lacking the Paf receptor (Ptafr(-/-)) and following inhibition of phosphatidylinositol-3 kinase or AKT. Inhibition of MDM2 caused a Trp53-dependent reduction in zygote development. Inbred strain embryos cultured from the zygote stage expressed less phosphorylated MDM2 than similar embryos collected from the uterus. The addition of Paf to the media caused increased phosphorylation of MDM2, and this was blocked by inhibitors of phosphatidylinositol-3 kinase and AKT. The study identifies trophic ligand signaling via the activation of phosphatidylinositol-3 kinase and AKT as a mechanism resulting in the activation of MDM2.

Restricting the growth of the embryo can cause adverse whole-of-life changes in an organism's homeostasis. Such adverse long-term consequences may occur even when growth restriction occurs only during the preimplantation period. The molecular basis for these long-term effects has not been defined, although an epigenetic mechanism is suspected. Some loci seem to be more sensitive to epigenetic perturbation than others, and the agouti viable yellow allele (A(vy)) is the best studied example of this. It has active (hypomethylated) and inactive (hypermethylated) epialleles. This study used the A(vy) model to show that growth restriction of preimplantation embryos, as provided by culture of zygotes, induced persistent epigenetic changes that resulted in altered postnatal phenotype. C57BL/6 A(vy)/a males were mated to ovulation-induced FVB/N females, and then either zygotes were collected and cultured for 96 h and the resulting blastocysts were transferred to pseudopregnant recipient females, blastocysts were collected from females and transferred without embryo culture, or pregnancy was allowed to proceed after mating without intervention. Culture was in a commercial human in vitro fertilization media. The proportion of pups expressing the active (hypomethylated) epiallele and yellow coat was significantly higher following zygote culture compared to embryos that were transferred without culture (P = 0.014) or natural matings (P < 0.001). There was no difference in expression of the active epiallele in pups resulting from embryo transfer (without culture) compared to natural matings. These results show for the first time that the preimplantation embryo's growth environment can affect the postnatal expression of a defined epigenetically sensitive allele.

Unlabelled: Limb salvage surgery is an effective procedure with a low risk of tumor recurrence. In an attempt to define the incidence of implant failure at 2, 5, and 10 years postoperatively, we retrospectively reviewed implant survival in a group (n = 105) of pediatric (< 18 years of age) and adult patients who were treated with distal femoral and/or proximal tibial implants for extremity tumors. Issues regarding the timing of failure, reason for failure, and whether pediatric patients had higher rates of failure were posed as secondary questions. The median followup was 57 months (1-235 months). Thirty-two (32/105, 31%) patients had 42 implant failures. The mean prosthesis Kaplan-Meier survivorship of the index group was 84% at 2 years, 73% at 5 years, and 59% at 10 years. Forty-seven percent of all failures occurred within 2 years postoperatively, and 69% occurred within 5 years postoperatively. Pediatric patients had a higher failure rate than adults (42% versus 24%). Aseptic loosening was the most common reason for failure (n = 18/32; 56%). The incidence of failure in tumor megaprostheses is similar to early published literature and the incidence of these failures is highest within the first 3 years., Level of Evidence: Therapeutic study, level IV.

The DNA deaminase family encompasses enzymes that have been highly conserved throughout vertebrate evolution and which display wide-ranging positive effects upon innate and adaptive immune system and development. Activation-induced cytidine deaminase was identified as a DNA mutator after its necessity in the successful development of high-affinity B cells via somatic hypermutation, class switch recombination, and gene conversion was determined. APOBEC3 exhibits the ability to deaminate retroviral first strand cDNA in a variety of viral infections, including HIV and hepatitis. Recent work has highlighted the potential importance of activation-induced cytidine deaminase (AID) and APOBEC1 in epigenetic reprogramming, and also the role that AID and the APOBECs may have in the development of cancer. In addition to the known activities of these members of the protein family, there are still other deaminases, such as APOBEC2, whose targets and functions are as yet unknown. This chapter provides the details of two assays that have proved to be invaluable in elucidating the exact specificities of deaminases both in vitro and in Escherichia coli. The application of these assays to future studies of the deaminase family will provide an indispensible tool in determining the potentially diverse functions of the remainder of this family of enzymes.

Background and Aims: Species' 2C-values (mass of DNA in G(1) phase 2n nuclei) vary by at least four orders of magnitude among seed plants. The 2C-value has been shown to be co-ordinated with a number of other species traits, and with environmental variables. A prediction that species 2C-values are negatively related to leaf life span (LL) and leaf mass per area (LMA) is tested. These leaf traits are components of a major dimension of ecological variation among plant species., Methods: Flow cytometry was used to measure the 2C-values for 41 Australian seed plant species, 40 of which were new to the literature. Where possible, LL and LMA data from the global literature were combined with 2C-values from our data set and online C-value databases., Key Results: Across all species, weak positive relationships were found between 2C-values and both LL and LMA; however, these did not reflect the relationships within either angiosperms or gymnosperms. Across 59 angiosperm species, there were weak negative relationships between 2C-values and both LL (r2 = 0.13, P = 0.005) and LMA (r2 = 0.15, P = 0.002). These relationships were the result of shifts to longer LL and greater LMA in woody compared with herbaceous growth forms, with no relationships present within growth forms. It was not possible to explain a positive relationship between 2C-values and LMA (r2 = 0.30, P = 0.024) across 17 gymnosperm species. The 2C-value was not related to LL or LMA either across species within orders (except for LMA among Pinales), or as radiation divergences in a model phylogeny., Conclusions: Gymnosperms appear to vary along a spectrum different from angiosperms. Among angiosperms, weak negative cross-species relationships were associated with growth form differences, and traced to a few divergences deep in the model phylogeny. These results suggest that among angiosperms, nuclear DNA content and leaf strategy are unrelated.

Epigenetic marking systems confer stability of gene expression during mammalian development. Genome-wide epigenetic reprogramming occurs at stages when developmental potency of cells changes. At fertilization, the paternal genome exchanges protamines for histones, undergoes DNA demethylation, and acquires histone modifications, whereas the maternal genome appears epigenetically more static. During preimplantation development, there is passive DNA demethylation and further reorganization of histone modifications. In blastocysts, embryonic and extraembryonic lineages first show different epigenetic marks. This epigenetic reprogramming is likely to be needed for totipotency, correct initiation of embryonic gene expression, and early lineage development in the embryo. Comparative work demonstrates reprogramming in all mammalian species analysed, but the extent and timing varies, consistent with notable differences between species during preimplantation development. Parental imprinting marks originate in sperm and oocytes and are generally protected from this genome-wide reprogramming. Early primordial germ cells possess imprinting marks similar to those of somatic cells. However, rapid DNA demethylation after midgestation erases these parental imprints, in preparation for sex-specific de novo methylation during gametogenesis. Aberrant reprogramming of somatic epigenetic marks after somatic cell nuclear transfer leads to epigenetic defects in cloned embryos and stem cells. Links between epigenetic marking systems appear to be developmentally regulated contributing to plasticity. A number of activities that confer epigenetic marks are firmly established, while for those that remove marks, particularly methylation, some interesting candidates have emerged recently which need thorough testing in vivo. A mechanistic understanding of reprogramming will be crucial for medical applications of stem cell technology.

DNA deaminases of the Aid/Apobec family convert cytosine into uracil and play key roles in acquired and innate immunity. The epigenetic modification by methylation of cytosine in CpG dinucleotides is also mutagenic, but this is thought to occur by spontaneous deamination. Here we show that Aid and Apobec1 are 5-methylcytosine deaminases resulting in a thymine base opposite a guanine. Their action can thus lead to C --> T transition mutations in methylated DNA, or in conjunction with repair of the T:G mismatch, to demethylation. The Aid and Apobec1 genes are located in a cluster of pluripotency genes including Nanog and Stella and are co-expressed with these genes in oocytes, embryonic germ cells, and embryonic stem cells. These results suggest that Aid and perhaps some of its family members may have roles in epigenetic reprogramming and cell plasticity. Transition in CpG dinucleotides is the most frequent mutation in human genetic diseases, and sequence context analysis of CpG transitions in the APC tumor suppressor gene suggests that DNA deaminases may play a significant role in tumor etiology.

The technique of cancellous impaction allografting with cement aims to reconstitute a bone-deficient proximal femur while also gaining stable fixation of the femoral prosthesis. Some reports of this technique imply it is a system, requiring not just an exacting surgical method, but also a particular implant design, the polished, double-tapered stem. Other series consider it a surgical technique, and have varied the femoral component design, the method of graft delivery, and other elements of the procedure. Our review evaluates the current literature, with the goal of beginning to ascertain whether published results suggest impaction grafting must be considered a system, requiring a particular stem design, or simply another means to achieve femoral reconstruction in the revision setting. The conclusive answer will require randomized, controlled clinical trials to evaluate particular elements of the procedure, and these studies have yet to be done. However, investigators have shown similarly good short-term to intermediate-term results with various femoral stems at numerous centers. Currently, femoral impaction allografting, whether as a system using particular implant designs or as a surgical technique, is an accepted alternative for revision of a failed femoral component, particularly when bone-stock deficiency is present.

Although in the proximal tibia the need for pin augmentation of cemented giant cell tumor defects depends on whether the defect is contained, there is controversy regarding the role for pins in the distal femur. The current study investigated whether Steinmann pin augmentation offers biomechanical advantages in cement reconstruction of contained defects of the lateral femoral condyle. Twelve pairs of human femurs were used. They were tested either with the bone intact, with a standardized contained defect in the lateral condyle, with a defect repaired with cement alone, and with a defect repaired with pins in the cement. Intact specimens had significantly higher load to failure than specimens with a defect. In the repaired specimens there were no significant differences in stiffness, peak load to failure, and energy to failure between the specimens repaired with just cement or augmented with pins. This study did not show any significant biomechanical advantage of this configuration of Steinmann pin reinforcement in cement for contained defects of the lateral femoral condyle.

Phenotypic variation that cannot be explained by genetic or environmental heterogeneity has intrigued geneticists for decades. The molecular basis of this phenomenon, however, is largely a mystery. Axin-fused (Axin(Fu)), first identified in 1937, is a classic example of a mammalian allele displaying extremely variable expression states. Here we demonstrate that the presence or absence of its characteristic phenotype, a kinked tail, correlates with differential DNA methylation at a retrotransposon within Axin(Fu) and identify mutant transcripts arising adjacent to the retrotransposon LTR that are likely to be causative of the phenotype. Furthermore, the epigenetic state at Axin(Fu) can be inherited transgenerationally after both maternal and paternal transmission. This is in contrast to epigenetic inheritance at the murine agouti-viable yellow (A(vy)) allele, which occurs through the female only. Unlike the egg, the sperm contributes very little (if any) cytoplasm to the zygote, and therefore paternal inheritance at Axin(Fu) argues against the possibility that the effects are due to cytoplasmic or metabolic influences. Consistent with the idea of transgenerational inheritance of epigenetic marks, we find that the methylation state of Axin(Fu) in mature sperm reflects the methylation state of the allele in the somatic tissue of the animal, suggesting that it does not undergo epigenetic reprogramming during gametogenesis. Finally, we show that epigenetic inheritance is influenced by strain background. These findings enable us to propose a model for transgenerational epigenetic inheritance in mammals.

It is well recognized that there is a surprising degree of phenotypic variation among genetically identical individuals, even when the environmental influences, in the strict sense of the word, are identical. Genetic textbooks acknowledge this fact and use different terms, such as 'intangible variation' or 'developmental noise', to describe it. We believe that this intangible variation results from the stochastic establishment of epigenetic modifications to the DNA nucleotide sequence. These modifications, which may involve cytosine methylation and chromatin remodelling, result in alterations in gene expression which, in turn, affects the phenotype of the organism. Recent evidence, from our work and that of others in mice, suggests that these epigenetic modifications, which in the past were thought to be cleared and reset on passage through the germline, may sometimes be inherited to the next generation. This is termed epigenetic inheritance, and while this process has been well recognized in plants, the recent findings in mice force us to consider the implications of this type of inheritance in mammals. At this stage we do not know how extensive this phenomenon is in humans, but it may well turn out to be the explanation for some diseases which appear to be sporadic or show only weak genetic linkage.

Epigenetic modifications that suppress gene activity in mammals are generally considered to be cleared in the germline, restoring totipotency of the genome. Here we report the germline inheritance of transcriptional silencing in mice, and reversion to activity after as many as three generations in the silent state. In a series of lines made with a LacZ transgene, one line exhibits variable expressivity: genotypically identical littermates have proportions of beta-Gal-positive erythrocytes that vary over at least four orders of magnitude, and in some offspring expression is completely silenced. The silent state of the transgene is inherited for multiple generations in the founder strain irrespective of the sex of the parent, implying maintenance of the epigenetic state through meiosis. Crosses of silenced mice with C57BL/6 mice result in reactivation of the transgene in approximately a third of F(1) littermates. The silencing involves a stochastic, all-or-none mechanism. Furthermore, silencing is transcriptional and correlates with methylation of the transgene as well as an inaccessible chromatin structure; these changes are reversed when expression is reactivated. This work supports the notion that silent genetic information in mammals can be inherited and later reactivated, and implies a mode of phenotypic inheritance that is less stable than Mendelian inheritance.

Epigenetic modifications have effects on phenotype, but they are generally considered to be cleared on passage through the germ line in mammals, so that only genetic traits are inherited. Here we describe the inheritance of an epigenetic modification at the agouti locus in mice. In viable yellow ( A(vy)/a) mice, transcription originating in an intra-cisternal A particle (IAP) retrotransposon inserted upstream of the agouti gene (A) causes ectopic expression of agouti protein, resulting in yellow fur, obesity, diabetes and increased susceptibility to tumours. The pleiotropic effects of ectopic agouti expression are presumably due to effects of the paracrine signal on other tissues. Avy mice display variable expressivity because they are epigenetic mosaics for activity of the retrotransposon: isogenic Avy mice have coats that vary in a continuous spectrum from full yellow, through variegated yellow/agouti, to full agouti (pseudoagouti). The distribution of phenotypes among offspring is related to the phenotype of the dam; when an A(vy) dam has the agouti phenotype, her offspring are more likely to be agouti. We demonstrate here that this maternal epigenetic effect is not the result of a maternally contributed environment. Rather, our data show that it results from incomplete erasure of an epigenetic modification when a silenced Avy allele is passed through the female germ line, with consequent inheritance of the epigenetic modification. Because retrotransposons are abundant in mammalian genomes, this type of inheritance may be common.

A program of repeated electrical (kindling-like) stimulation of the medial preoptic area (MPOA) or the medial amygdala (MedAmyg) on maternal and other behaviors were investigated. Stimulation was applied daily for 14 days (or until a stage 3 motor seizure was observed) using 2 s trains of biphasic square wave pulses at 60 Hz, 1 ms duration and 300-500 microA. Confirmation of afterdischarge using these parametres was established. In the first experiment, maternally experienced (but not post-partum) MedAmyg stimulated animals became maternal more slowly than did MedAmyg not stimulated animals or than MPOA stimulated animals. In the second experiment, virgin animals were used. MPOA stimulation enhanced the female's preference for pup associated environments in the conditioned place preference (CPP) paradigm. MedAmyg stimulation had no effect on CPP performance, but produced a decreased preference for pup odors in a modified hole board test and increased 'anxiety' in the open field. These results confirm that the MPOA and the MedAmyg are involved in facilitating and attenuating maternal responsiveness and related (precursor?) behaviors, respectively. It appears that chronic (kindling-like) stimulation of these neural substrates enhances their functions.

The effects on rodent anxiety of kindling in the medial or basolateral amygdaloid nuclei in each hemisphere were examined. Anxiety was measured using the hole board and elevated plus maze tests. The animals were kindled in medial or basolateral amygdalas, of either the left or right hemisphere. Controls had electrodes implanted in comparable areas, but were not kindled. Analysis of electrode location showed that some animals were kindled in amygdaloid nuclei other than medial or basolateral amygdala. These animals were labelled outliers. Kindling of the medial/basolateral amygdala in the left hemisphere decreased anxiety for at least 1 week after the last kindled seizure. Right hemisphere medial/basolateral kindling tended to increase anxiety. Outlier-kindled rats were less anxious than their controls regardless of hemisphere 1 week after their last kindled seizure. Clear anxiogenic effects were not likely seen in the right hemisphere in this study because of the electrode locations. The degree of anxiety following kindling was correlated with electrode location in the anterior-posterior plane. More anterior foci in the amygdala were associated with more anxiety. More posterior amygdala foci were associated with less anxiety. These findings point to the importance of kindled focus in the amygdala for behavioral effect. Future research should carefully control the location of kindled foci when investigating effects of amygdala kindling on anxiety and other behaviors.

The role of perioral and ventral-trunk somatosensory stimulation from pups mediating the initial expression of maternal behavior and its long-term retention 8 days later, was investigated. Six groups of female rats were permitted to physically interact with four 1-5-day-old foster pups for 1 h, 36 h after Cesarean delivery on gestation day 21. Prior to this maternal experience, dams were subjected to: reduced cutaneous rostral snout sensitivity (anaptia) by injection of lidocaine into the mystacial pads; reduced ventral-trunk sensations by occlusion of the entire ventrum with a full spandex jacket; both manipulations; or control manipulations. Additional groups of females not receiving a maternal experience (inexperienced) also received the somatosensory deprivation or control manipulations. During retention testing, rats in the singly manipulated experienced groups exhibited reduced latencies to become maternal in comparison to their inexperienced counterparts (approximately 3 days vs. 8 days). However, rats previously rendered both anaptic and ventrally-occluded responded like inexperienced rats in showing a long latency to become maternal (8 days). Thus, reduction of either perioral or ventral somatosensory contact from pups did not block the maternal experience effect, but reduction of both of these inputs did.

Two types of colonies of laboratory mice were employed; hierarchically organized ones formed by placing five unfamiliar 8-week-old mice in a cage together and amicably organized colonies in which four male litter mates were kept together throughout the whole period of the experiment. During a 21-day pre-experimental period intra-colony aggressive behaviour was recorded. A dominant animal and ranked subordinates occurred in every hierarchical colony, whilst no aggression was recorded in any of the amicable colonies. During a 25-day period unfamiliar adult male or female mice were introduced daily into the amicable or hierarchical colonies for 10 min. In a third experiment juvenile mice 17, 24, 31 or 38 days old were introduced into hierarchically organized colonies during a 20-day period. In all hierarchical colonies the stranger was attacked irrespective of sex and age; the majority of attacks were carried out by the dominant mouse. Aggression by the dominant declined exponentially throughout the experimental period and regression analyses compared the different data. Unfamiliar adult females were the recipients of fewer attacks than unfamiliar adult males and the age of juvenile strangers was found to be an important factor. Amicably organized mice initially did not attack strangers, but over a period of 25 days the number of attacks on unfamiliar males gradually increased.