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The free-living, simultaneously hermaphroditic flatworms of the genus Macrostomum are increasingly used as model systems in various contexts. In particular, Macrostomum lignano, the only species of this group with a published genome assembly, has emerged as a model for the study of regeneration, reproduction, and stem-cell function. However, challenges have emerged due to M. lignano being a hidden polyploid, having recently undergone whole-genome duplication and chromosome fusion events. This complex genome architecture presents a significant roadblock to the application of many modern genetic tools. Hence, additional genomic resources for this genus are needed. Here, we present such resources for Macrostomum cliftonense and Macrostomum hystrix, which represent the contrasting mating behaviors of reciprocal copulation and hypodermic insemination found in the genus. We use a combination of PacBio long-read sequencing and Illumina shot-gun sequencing, along with several RNA-Seq data sets, to assemble and annotate highly contiguous genomes for both species. The assemblies span ~227 and ~220 Mb and are represented by 399 and 42 contigs for M. cliftonense and M. hystrix, respectively. Furthermore, high BUSCO completeness (~84-85%), low BUSCO duplication rates (8.3-6.2%), and low k-mer multiplicity indicate that these assemblies do not suffer from the same assembly ambiguities of the M. lignano genome assembly, which can be attributed to the complex karyology of this species. We also show that these resources, in combination with the prior resources from M. lignano, offer an excellent foundation for comparative genomic research in this group of organisms. [ABSTRACT FROM AUTHOR]

The Y chromosome is theorized to facilitate evolution of sexual dimorphism by accumulating sexually antagonistic loci, but empirical support is scarce. Due to the lack of recombination, Y chromosomes are prone to degenerative processes, which poses a constraint on their adaptive potential. Yet, in the seed beetle, Callosobruchus maculatus segregating Y linked variation affects male body size and thereby sexual size dimorphism (SSD). Here, we assemble C. maculatus sex chromosome sequences and identify molecular differences associated with Y-linked SSD variation. The assembled Y chromosome is largely euchromatic and contains over 400 genes, many of which are ampliconic with a mixed autosomal and X chromosome ancestry. Functional annotation suggests that the Y chromosome plays important roles in males beyond primary reproductive functions. Crucially, we find that, besides an autosomal copy of the gene target of rapamycin (TOR), males carry an additional TOR copy on the Y chromosome. TOR is a conserved regulator of growth across taxa, and our results suggest that a Y-linked TOR provides a male specific opportunity to alter body size. A comparison of Y haplotypes associated with male size difference uncovers a copy number variation for TOR , where the haplotype associated with decreased male size, and thereby increased sexual dimorphism, has two additional TOR copies. This suggests that sexual conflict over growth has been mitigated by autosome to Y translocation of TOR followed by gene duplications. Our results reveal that despite of suppressed recombination, the Y chromosome can harbor adaptive potential as a male-limited supergene. [ABSTRACT FROM AUTHOR]

The patterns of reproductive timing and senescence vary within and across species owing to differences in reproductive strategies, but our understanding of the molecular underpinnings of such variation is incomplete. This is perhaps particularly true for sex differences. We investigated the evolution of sex-specific gene expression associated with life history divergence in replicated populations of the seed beetle Acanthoscelides obtectus , experimentally evolving under (E)arly or (L)ate life reproduction for >200 generations which has resulted in strongly divergent life histories. We detected 1,646 genes that were differentially expressed in E and L lines, consistent with a highly polygenic basis of life history evolution. Only 30% of differentially expressed genes were similarly affected in males and females. The evolution of long life was associated with significantly reduced sex differences in expression, especially in non-reproductive tissues. The expression differences were overall more pronounced in females, in accordance with their greater phenotypic divergence in lifespan. Functional enrichment analysis revealed differences between E and L beetles in gene categories previously implicated in aging, such as mitochondrial function and defense response. The results show that divergent life history evolution can be associated with profound changes in gene expression that alter the transcriptome in a sex-specific way, highlighting the importance of understanding the mechanisms of aging in each sex. [ABSTRACT FROM AUTHOR]

Gene repertoire turnover is a characteristic of genome evolution. However, we lack well‐replicated analyses of presence/absence patterns associated with different selection contexts. Here, we study ∼100 transcriptome assemblies across Macrostomum, a genus of simultaneously hermaphroditic flatworms exhibiting multiple convergent shifts in mating strategy and associated reproductive morphologies. Many species mate reciprocally, with partners donating and receiving sperm at the same time. Other species convergently evolved to mate by hypodermic injection of sperm into the partner. We find that for orthologous transcripts annotated as expressed in the body region containing the testes, sequences from hypodermically inseminating species diverge more rapidly from the model species, Macrostomum lignano, and have a lower probability of being observed in other species. For other annotation categories, simpler models with a constant rate of similarity decay with increasing genetic distance from M. lignano match the observed patterns well. Thus, faster rates of sequence evolution for hypodermically inseminating species in testis‐region genes result in higher rates of homology detection failure, yielding a signal of rapid evolution in sequence presence/absence patterns. Our results highlight the utility of considering appropriate null models for unobserved genes, as well as associating patterns of gene presence/absence with replicated evolutionary events in a phylogenetic context. [ABSTRACT FROM AUTHOR]

Increasing temperature and thermal variability generate profound selection on populations. Given the fast rate of environmental change, understanding the role of plasticity and genetic adaptation in response to increasing temperatures is critical. This may be especially true for thermal effects on reproductive traits in which thermal fertility limits at high temperatures may be lower than for survival traits. Consequences of changing environments during development on adult phenotypes may be particularly problematic for core traits such as reproduction that begin early in development. Here we examine the consequences of developmental thermal plasticity on subsequent adult reproductive traits and its genetic basis.We used a panel of Drosophila melanogaster (the Drosophila Genetic Reference Panel; DGRP) in which male fertility performance was previously defined as either showing relatively little (status = 'high'‐performing lines) or substantial ('low'‐performing lines) decline when exposed to increasing developmental temperatures. We used a thermal reaction norm approach to quantify variation in the consequences of developmental thermal plasticity on multiple adult reproductive traits, including sex‐specific responses, and to identify candidate genes underlying such variation.Developmental thermal stress impacted the means and thermal reaction norms of all reproductive traits except offspring sex ratio. Mating success declined as temperature increased with no difference between high and low lines, whereas increasing temperature resulted in declines for both male and female fertility and productivity but depended on line status. Fertility and offspring number were positively correlated within and between the sexes across lines, but males were more affected than females.We identified 933 SNPs with significant evolved genetic differentiation between high and low lines. In all, 54 of these lie within genomic windows of overall high differentiation, have significant effects of genotype on the male thermal reaction norm for productivity and are associated with 16 genes enriched for phenotypes affecting reproduction, stress responses and autophagy in Drosophila and other organisms.Our results illustrate considerable plasticity in male thermal limits on several reproductive traits following development at high temperature, and we identify differentiated loci with relevant phenotypic effects that may contribute to this population variation. While our work is on a single population, phenotypic results align with an increasing number of studies demonstrating the potential for stronger selection of thermal stress on reproductive traits, particularly in males. Such large fitness costs may have both short‐ and long‐term consequences for the evolution of populations in response to a warming world. [ABSTRACT FROM AUTHOR]

Sexual selection drives the evolution of many striking behaviors and morphologies and should leave signatures of selection at loci underlying these phenotypes. However, although loci thought to be under sexual selection often evolve rapidly, few studies have contrasted rates of molecular sequence evolution at such loci across lineages with different sexual selection contexts. Furthermore, work has focused on separate sexed animals, neglecting alternative sexual systems. We investigate rates of molecular sequence evolution in hermaphroditic flatworms of the genus Macrostomum. Specifically, we compare species that exhibit contrasting sperm morphologies, strongly associated with multiple convergent shifts in the mating strategy, reflecting different sexual selection contexts. Species donating and receiving sperm in every mating have sperm with bristles, likely to prevent sperm removal. Meanwhile, species that hypodermically inject sperm lack bristles, potentially as an adaptation to the environment experienced by hypodermic sperm. Combining functional annotations from the model, Macrostomum lignano , with transcriptomes from 93 congeners, we find genus-wide faster sequence evolution in reproduction-related versus ubiquitously expressed genes, consistent with stronger sexual selection on the former. Additionally, species with hypodermic sperm morphologies had elevated molecular sequence evolution, regardless of a gene's functional annotation. These genome-wide patterns suggest reduced selection efficiency following shifts to hypodermic mating, possibly due to higher selfing rates in these species. Moreover, we find little evidence for convergent amino acid changes across species. Our work not only shows that reproduction-related genes evolve rapidly also in hermaphroditic animals, but also that well-replicated contrasts of different sexual selection contexts can reveal underappreciated genome-wide effects. [ABSTRACT FROM AUTHOR]

Detecting signatures of ecological adaptation in comparative genomics is challenging, but analysing population samples with characterised geographic distributions, such as clinal variation, can help identify genes showing covariation with important ecological variation. Here, we analysed patterns of geographic variation in the cold‐adapted species Drosophilamontana across phenotypes, genotypes and environmental conditions and tested for signatures of cold adaptation in population genomic divergence. We first derived the climatic variables associated with the geographic distribution of 24 populations across two continents to trace the scale of environmental variation experienced by the species, and measured variation in the cold tolerance of the flies of six populations from different geographic contexts. We then performed pooled whole genome sequencing of these six populations, and used Bayesian methods to identify SNPs where genetic differentiation is associated with both climatic variables and the population phenotypic measurements, while controlling for effects of demography and population structure. The top candidate SNPs were enriched on the X and fourth chromosomes, and they also lay near genes implicated in other studies of cold tolerance and population divergence in this species and its close relatives. We conclude that ecological adaptation has contributed to the divergence of D. montana populations throughout the genome and in particular on the X and fourth chromosomes, which also showed highest interpopulation FST. This study demonstrates that ecological selection can drive genomic divergence at different scales, from candidate genes to chromosome‐wide effects. [ABSTRACT FROM AUTHOR]

Comparative genomics has contributed to the growing evidence that sexual selection is an important component of evolutionary divergence and speciation. Divergence by sexual selection is implicated in faster rates of divergence of the X chromosome and of genes thought to underlie sexually selected traits, including genes that are sex biased in expression. However, accurately inferring the relative importance of complex and interacting forms of natural selection, demography, and neutral processes that occurred in the evolutionary past is challenging. Experimental evolution provides an opportunity to apply controlled treatments for multiple generations and examine the consequent genomic divergence. Here, we altered sexual selection intensity, elevating sexual selection in polyandrous lines and eliminating it in monogamous lines, and examined patterns of allele frequency divergence in the genome of Drosophila pseudoobscura after more than 160 generations of experimental evolution. Divergence is not uniform across the genome but concentrated in "islands," many of which contain candidate genes implicated in mating behaviors and other sexually selected phenotypes. These are more often seen on the X chromosome, which also shows greater divergence in FST than neutral expectations. There are characteristic signatures of selection seen in these regions, with lower diversity on the X chromosome than the autosomes, and differences in diversity on the autosomes between selection regimes. Reduced Tajima's D within some of the divergent regions may imply that selective sweeps have occurred, despite considerable recombination. These changes are associated with both differential gene expression between the lines and sex‐biased gene expression within the lines. Our results are very similar to those thought to implicate sexual selection in divergence between species and natural populations, and hence provide experimental support for the likely role of sexual selection in driving such types of genetic divergence, but also illustrate how variable outcomes can be for different genomic regions. [ABSTRACT FROM AUTHOR]

Very few animals habitually manufacture and use tools. It has been suggested that advanced tool behaviour co‐evolves with a suite of behavioural, morphological and life history traits. In fact, there are indications for such an adaptive complex in tool‐using crows (genus Corvus species). Here, we sequenced the genomes of two habitually tool‐using and ten non‐tool‐using crow species to search for genomic signatures associated with a tool‐using lifestyle. Using comparative genomic and population genetic approaches, we screened for signals of selection in protein‐coding genes in the tool‐using New Caledonian and Hawaiian crows. While we detected signals of recent selection in New Caledonian crows near genes associated with bill morphology, our data indicate that genetic changes in these two lineages are surprisingly subtle, with little evidence at present for convergence. We explore the biological explanations for these findings, such as the relative roles of gene regulation and protein‐coding changes, as well as the possibility that statistical power to detect selection in recently diverged lineages may have been insufficient. Our study contributes to a growing body of literature aiming to decipher the genetic basis of recently evolved complex behaviour. [ABSTRACT FROM AUTHOR]

Genetic variation is the fuel of evolution, with standing genetic variation especially important for short-term evolution and local adaptation. To date, studies of spatiotemporal patterns of genetic variation in natural populations have been challenging, as comprehensive sampling is logistically difficult, and sequencing of entire populations costly. Here, we address these issues using a collaborative approach, sequencing 48 pooled population samples from 32 locations, and perform the first continent-wide genomic analysis of genetic variation in European Drosophila melanogaster. Our analyses uncover longitudinal population structure, provide evidence for continent-wide selective sweeps, identify candidate genes for local climate adaptation, and document clines in chromosomal inversion and transposable element frequencies. We also characterize variation among populations in the composition of the fly microbiome, and identify five new DNA viruses in our samples. [ABSTRACT FROM AUTHOR]

Theory predicts that deleterious mutations accumulate more readily in small populations. As a consequence, mutation load is expected to be elevated in species where life-history strategies and geographic or historical contingencies reduce the number of reproducing individuals. Yet, few studies have empirically tested this prediction using genome-wide data in a comparative framework. We collected whole-genome sequencing data for 147 individuals across seven crow species (Corvus spp.). For each species, we estimated the distribution of fitness effects of deleterious mutations and compared it with proxies of the effective population size N e. Island species with comparatively smaller geographic range sizes had a significantly increased mutation load. These results support the view that small populations have an elevated risk of mutational meltdown, which may contribute to the higher extinction rates observed in island species. [ABSTRACT FROM AUTHOR]

The genomes of species that are ecological specialists will likely contain signatures of genomic adaptation to their niche. However, distinguishing genes related to ecological specialism from other sources of selection and more random changes is a challenge. Here, we describe the genome of Drosophila montana, which is the most extremely cold-adapted Drosophila species known. We use branch tests to identify genes showing accelerated divergence in contrasts between cold- and warm-adapted species and identify about 250 genes that show differences, possibly driven by a lower synonymous substitution rate in cold-adapted species. We also look for evidence of accelerated divergence between D. montana and D. virilis, a previously sequenced relative, but do not find strong evidence for divergent selection on coding sequence variation. Divergent genes are involved in a variety of functions, including cuticular and olfactory processes. Finally, we also resequenced three populations of D. montana from across its ecological and geographic range. Outlier loci were more likely to be found on the X chromosome and there was a greater than expected overlap between population outliers and those genes implicated in cold adaptation between Drosophila species, implying some continuity of selective process at these different evolutionary scales. [ABSTRACT FROM AUTHOR]

With increasing application of pooled-sequencing approaches to population genomics robust methods are needed to accurately quantify allele frequency differences between populations. Identifying consistent differences across stratified populations can allow us to detect genomic regions under selection and that differ between populations with different histories or attributes. Current popular statistical tests are easily implemented in widely available software tools which make them simple for researchers to apply. However, there are potential problems with the way such tests are used, which means that underlying assumptions about the data are frequently violated., These problems are highlighted by simulation of simple but realistic population genetic models of neutral evolution and the performance of different tests are assessed. We present alternative tests (including Generalised Linear Models [ GLMs] with quasibinomial error structure) with attractive properties for the analysis of allele frequency differences and re-analyse a published dataset., The simulations show that common statistical tests for consistent allele frequency differences perform poorly, with high false positive rates. Applying tests that do not confound heterogeneity and main effects significantly improves inference. Variation in sequencing coverage likely produces many false positives and re-scaling allele frequencies to counts out of a common value or an effective sample size reduces this effect., Many researchers are interested in identifying allele frequencies that vary consistently across replicates to identify loci underlying phenotypic responses to selection or natural variation in phenotypes. Popular methods that have been suggested for this task perform poorly in simulations. Overall, quasibinomial GLMs perform better and also have the attractive feature of allowing correction for multiple testing by standard procedures and are easily extended to other designs. [ABSTRACT FROM AUTHOR]

Testing for conserved and novel mechanisms underlying phenotypic evolution requires a diversity of genomes available for comparison spanning multiple independent lineages. For example, complex social behavior in insects has been investigated primarily with eusocial lineages, nearly all of which are Hymenoptera. If conserved genomic influences on sociality do exist, we need data from a wider range of taxa that also vary in their levels of sociality. Here, we present the assembled and annotated genome of the subsocial beetle Nicrophorus vespilloides, a species long used to investigate evolutionary questions of complex social behavior. We used this genome to address two questions. First, do aspects of life history, such as using a carcass to breed, predict overlap in gene models more strongly than phylogeny? We found that the overlap in gene models was similar between N. vespilloides and all other insect groups regardless of life history. Second, like other insects with highly developed social behavior but unlike other beetles, does N. vespilloides have DNA methylation? We found strong evidence for an active DNA methylation system. The distribution of meth-ylation was similar to other insects with exons having the most methylated CpGs. Methylation status appears highly conserved; 85% of the methylated genes in N. vespilloides are also methylated in the hymentopteran Nasonia vitripennis. The addition of this genome adds a coleopteran resource to answer questions about the evolution and mechanistic basis of sociality and to address questions about the potential role of methylation in social behavior. [ABSTRACT FROM AUTHOR]

Long noncoding RNAs (lncRNAs) are one of themost intensively studied groups of noncoding elements.Debate continues overwhat proportion of lncRNAs are functional ormerely represent transcriptional noise. Although characterization of individual lncRNAs has identified approximately 200 functional loci across the Eukarya, general surveys have found only modest or no evidence of long-term evolutionary conservation. Although this lack of conservation suggests that most lncRNAs are nonfunctional, the possibility remains thatsomerepresent recent evolutionary innovations.Weexaminerecent selectionpressuresactingonlncRNAsinmousepopulations. Wecomparepatternsof within-species nucleotide variation at approximately10,000 lncRNAloci ina cohort of the wild housemouse, Mus musculus castaneus, with between-species nucleotide divergence from the rat (Rattus norvegicus). Loci under selective constraint are expected to show reduced nucleotide diversity and divergence. We find limited evidence of sequence conservation comparedwithputativelyneutrally evolving ancestral repeats (ARs).Comparisonsof sequence diversity anddivergence betweenARs, protein-coding (PC) exons and lncRNAs, and the associated flanking regions, show weak, but significantly lower levels of sequence diversity and divergence at lncRNAs compared with ARs. lncRNAs conserved deep in the vertebrate phylogeny show lower withinspecies sequence diversity than lncRNAs in general. A set of 74 functionally characterized lncRNAs show levels of diversity and divergence comparable to PC exons, suggesting that these lncRNAs are under substantial selective constraints. Our results suggest that, inmouse populations,most lncRNA loci evolve at rates similar to ARs,whereas older lncRNAs tend to show signals of selection similar to PC genes. [ABSTRACT FROM AUTHOR]

The capacity of salbutamol 0.3 mg (SAL), disodium cromoglycate 3 mg (DSCG), a combination (SAL + DSCG), and ipratropium bromide 80 ug (IB), all given as metered aerosols to prevent exercise-induced (EIA), was compared with that of a placebo, a peroral lactose pill. Seven children participated, having reproducible EIA provoked by running on a treadmill at a heart rate of 170 sustained for 6 min. FEV1 and volume of trapped gas (VTG), defined as the air volume released during rebreathing oxygen with maximum breaths at the end of a multiple breath nitrogen wash out, were used as tests of spasm in large and small airways. SAL and SAL + DSCG offered complete protection in large and small airways. DSCG and IB prevented EIA in large airways (FEV1) to 95%, but only to about 50% in small airways (VTG). SAL or SAL + DSCG gave significantly better protection (FEV1 and VTG) than DSCG and IB (P< 0.01). Differences between DSCG and IB were not statistically significant. DSCG or propellant caused significant irritation and spasm in small airways (VTG) before exercise. Most subjects seemed to obtain satisfactory protection against EIA by β2-agonists. [ABSTRACT FROM AUTHOR]

`The Present study compared the bronchodilating effect of inhalation from the Turbuhaler® (0.5 mg terbutaline &time; 2) with the effect of inhalation from the chlorofluorocarbon (CFC) inhaler (0.25 mg terbutaline &time; 4) in children aged 9-17 years with reproducible, exercise-induced asthma (EIA). The treatments were given on two occasions 5 min apart (terbutaline 0.5 mg +0.5 mg). The study was performed as a double-blind double-dummy, and Placebo-controlled trial in 12 asthmatic children. The study was conducted on three separate days. The bronchoconstriction was induced by steady running' on a treadmill, Forced expiratory volume in 1 s (FEV1.0) vital capacity (VC), and volume of trapped gas (VTG) were measured before and after the exercise test arid after treatment The study showed that the same amount of terbutaline inhaled from the Turbuhaler or from a CFC inhaler is equally effective for reversing EIA, and that the Turbuhaler is possibly more effective for treating spasm in small airways. [ABSTRACT FROM AUTHOR]

The prevalence and diversity of female ornaments pose a challenge to evolutionary theory because males should prefer mates that spend resources on offspring rather than on ornaments. Among dance flies, there is extraordinary variation in sexual dimorphism. Females of many species have conspicuous ornaments (leg scales and inflatable abdominal sacs). Meanwhile, males of some species have exaggerated regions of their eyes with larger ommatidial facets that allow for regionally elevated photosensitivity and/or acuity. Here, we conduct a comparative study of these traits using both species descriptions available from the literature, as well as quantitative measures of eyes and ornaments from wild-caught flies. We show a conspicuous covariance across species between exaggerated male dorsal eye regions and the extent of female ornaments: species with highly ornamented females have males with more exaggerated eyes. We discuss this pattern in the context of competing hypotheses for the evolution of these traits and propose a plausible role for sexually antagonistic coevolution., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Society for the Study of Evolution (SSE).)

Background: Breast cancer is the most common cancer in women. Cancer cells can persist in a prolonged dormant state for years without any clinical evidence of disease creating an urgent need to better understand the molecular mechanisms leading to relapse. This study aimed to identify extracellular matrix (ECM) components associated with hypoxia-induced breast cancer dormancy. The effects of selected ECM proteins on breast cancer cell proliferation were analyzed, along with their correlation with established prognostic markers in human breast cancer tissue., Materials and Methods: Screening of extracellular matrix proteins was performed in hypoxia-induced dormant MCF-7 breast cancer cells. Proliferation of MCF-7 cells in vitro was subsequently determined in the presence of recombinant ColVII. Adipose tissue-derived mesenchymal stem cells (AdMSCs) subpopulation overexpressing ColVII were indirectly isolated by ColVII receptor integrin-α6 specific antibodies. AdMSCs- MCF-7 3D spheroid cultures were generated to model solid tumour conditions. In addition, the association between ColVII and various prognostic markers was evaluated in clinical samples of human breast cancer tissue., Results: Dormant MCF-7 cells showed an elevated expression of ColVII while MCF-7 cells cultured on ColVII exhibited reduced proliferation in vitro. In AdMSCs-MCF-7 3D spheroids, a reduced proliferation of MCF-7 cells was observed in Int-α6 + / ColVII high compared with Int-α6 -/ ColVII low AdMSCs spheroids. In human tissue, high ColVII expression correlated to several positive prognostic markers. Staining for Cytokeratin-5 revealed that ColVII high -expressing cells were predominantly myoepithelial cells., Conclusion: ColVII is associated with reduced proliferation of breast cancer cells in vitro. ColVII is strongly expressed in myoepithelial cells and in breast cancer tissue the high ColVII expression correlates with several well-known positive prognostic markers, highlighting its potential as a prognostic marker in breast cancer., (© 2024. The Author(s).)

Background: Soft tissue defects or periprosthetic infections after total knee arthroplasty (TKA) are severe complications that may lead to loss of the arthroplasty or the limb. Reconstructions with medial gastrocnemius flaps (MGF) are occasionally used to provide soft tissue coverage around the knee., Aims: The study aimed to establish the rate of implant survivorship after MGF reconstruction for soft tissue coverage in the treatment of exposed or infected TKA and to establish functional outcome., Methods: A retrospective analysis was performed on all patients who received soft tissue coverage with an MGF of an exposed or infected TKA between 2000 and 2017 at the Department of Hand and Plastic Surgery at Umeå University Hospital. The outcomes were implant survivorship and patient-reported outcome measures (PROMs) using the five-level EQ-5D version and The Knee Injury and Osteoarthritis Outcome Score., Results: Forty-seven patients (mean age = 67 years, 30 women) were included. The mean time between flap coverage and follow-up was 6.7 (±3.4) years. Implant survivorship was observed in 28 of 47 (59.6%) patients at follow-up. Flap failure was rare, with only 3 of 47 (6.4%) cases. Of the 20 patients who answered the PROMs, 10 of 20 experienced moderate to severe pain or discomfort., Conclusions: Due to unfavorable underlying conditions, MGF reconstruction after TKA is often associated with a compromised functional outcome. Because donor site morbidity is limited and flap failure is unusual, the procedure can be considered prophylactically in a small subset of patients with risk factors to prevent soft tissue defects and periprosthetic joint infection.

Background: Although autologous fat grafting is considered a successful method for the management of contour deformities, the fat graft could potentially induce cancer reappearance by fueling dormant breast cancer cells. Our aim was to characterize the role of adipose-derived stem cells on active and dormant breast cancer cell growth., Methods: Cobalt chloride was used to induce dormancy in MCF-7 cancer cells. Proliferation of active and dormant cancer cells was determined in the presence of adipose-derived stem cells. A proteome array was used to detect cancer-related protein expression in the cell-conditioned medium. The migration of cancer cells was measured in response to conditioned medium from the adipose-derived stem cells., Results: The adipose-derived stem cells showed variable effects on active MCF-7 cells growth and inhibited MCF-7 proliferation after the withdrawal of cobalt chloride. Of the 84 different proteins measured in the conditioned medium, only tenascin-C was differentially expressed in the co-cultures. MCF-7 cells alone did not express tenascin-C, whereas co-cultures between MCF-7 and adipose-derived stem cells expressed more tenascin-C versus adipose-derived stem cells alone. The conditioned medium from co-cultures significantly increased the migration of the cancer cells., Conclusions: Adipose-derived stem cells themselves neither increased the growth or migration of cancer cells, suggesting that autologous fat grafting may be oncologically safe if reconstruction is postponed until there is no evidence of active disease. However, interactions between adipose-derived stem cells and MCF-7 cancer cells could potentially lead to the production of factors, which further promote cancer cell migration., Competing Interests: Declaration of Competing Interest None of the authors has a financial interest in any of the products, devices, or drugs mentioned in this manuscript., (Copyright © 2023 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.)

Background: There are no published international consensus or guideline documents regarding appropriate medical follow-up for women with hereditary increased risk of breast cancer who opt for prophylactic mastectomy. Moreover, it is not known whether breast magnetic resonance imaging (MRI) performed after a prophylactic mastectomy is a reproducible method for evaluating whether clinically relevant amounts of residual glandular tissue remains., Purpose: To evaluate the inter- and intra-observer agreement on detecting residual glandular tissue with MRI., Material and Methods: In total, 40 women previously operated with prophylactic mastectomy underwent MRI and two breast radiologists (R1 and R2) independently assessed the presence of residual glandular tissue. Inter- and intra-rater agreements were assessed using Cohen's kappa (k)., Results: Residual glandular tissue was found in 69 of 248 quadrants (27.8%) and 32 of 62 breasts (51.6%) by R1 and 77 of 248 quadrants (31.1%) and 35 of 62 breasts (56.5%) by R2. The interrater agreement was observed to be moderate (k = 0.554) and the intra-rater agreement was observed to be substantial (k = 0.623)., Conclusion: In conclusion, the inter-and intra-rater observer agreement in regard to detection of residual glandular tissue was not excellent, which would be desirable for a method considered reproducible enough to be used as a surveillance tool after the surgical procedure in order to ensure that there is no relevant residual glandular tissue remaining warranting further follow-up. More research is needed, as well as establishment of precise protocols, before using the method in risk assessment of remaining glandular tissue and breast cancer risk.

Background: Women with an increased hereditary risk of breast cancer can undergo risk-reducing prophylactic mastectomy. However, there is a balance between how much subcutaneous tissue should be resected to achieve maximal reduction of glandular tissue, while leaving viable skin flaps., Methods: Forty-five women previously operated with prophylactic mastectomy underwent magnetic resonance tomography (MRT) and ultrasound (US) to investigate the correlation between skin flap thickness and residual glandular tissue. Residual glandular tissue was documented as being present or not present, but not quantified, as the amount of residual glandular tissue in many cases was considered too small to make reliable volume quantifications with available tools. Since a mastectomy skin flap thickness of 5 mm is discussed as an oncologically safe thickness in the literature, this was used as a cut-off., Results: Following prophylactic mastectomy, residual glandular tissue was detected in 39.3% of all breasts and 27.9% of all the breast quadrants examined by MRT, and 44.1% of all breasts and 21.7% of all the breast quadrants examined by US. Residual glandular tissue was detected in 6.9% of the quadrants in skin flaps ≤ 5 mm and in 37.5% of the quadrants in skin flaps > 5 mm (OR 3.07; CI = 1.41-6.67; p = 0.005). Furthermore, residual glandular tissue increased significantly already when the skin flap thickness exceeded 7 mm., Conclusions: This study highlights that complete removal of glandular breast tissue during a mastectomy is difficult and suggests that this is an unattainable goal. We demonstrate that residual glandular tissue is significantly higher in skin flaps > 5 mm in comparison to skin flaps ≤ 5 mm, and that residual glandular tissue increases significantly already when the flap thickness exceeds 7 mm., Competing Interests: Declaration of Competing Interest None of the authors has a financial interest in any of the products, devices or drugs mentioned in this manuscript., (Copyright © 2022 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.)

Introduction: Water jet-assisted liposuction has gained popularity due to favourable fat grafting outcomes. In this study, we compared stem cells obtained from fat isolated with manual or the water jet-assisted procedure., Methods: Liposuction of abdominal fat was performed using the two methods on each donor (n = 10). Aspirate samples were collagenase digested and the isolated cells seeded in vitro prior to proliferation, adipogenic differentiation and angiogenic activity analyses., Results: Cells from either procedure proliferated at similar rates and exhibited a similar colony-forming ability. The cells expressed stem cell markers CD73, CD90 and CD105. In the water jet cell preparations, there were higher numbers of cells expressing CD146. Robust adipogenic differentiation was observed in cultures expanded from both manual and water jet lipoaspirates. Gene analysis showed higher expression of the adipocyte markers aP2 and GLUT4 in the adipocyte-differentiated water jet cell preparations, and ELISA indicated increased secretion of adiponectin from these cells. Both cell groups expressed vasculogenic factors and the water jet cells promoted the highest levels of in vitro angiogenesis. Given these positive results, we further characterised the water jet cells when prepared using an automated closed cell processing unit, the Sepax-2 system (Cytiva). The growth and stem cell properties of the Sepax-processed cells were similar to the standard centrifugation protocol, but there was evidence for greater adipogenic differentiation in the Sepax-processed cells., Conclusions: Water jet lipoaspirates yield cells with high adipogenic potential and angiogenic activity, which may be beneficial for use in cell-assisted lipotransfers., (Copyright © 2021 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.)

Background: Women with an increased hereditary risk of breast cancer can undergo prophylactic mastectomy (PM), which provides a significant, but not total, risk reduction. There is an ongoing discussion about how much skin and subcutaneous tissue should be resected to perform an adequate PM while leaving viable skin flaps., Methods: Forty-five women who had undergone PM were examined with magnetic resonance tomography (MRT), ultrasound (US) and clinical examination (CE) by a plastic surgeon and a general surgeon to estimate skin flap thickness., Results: The estimated mean skin flap thickness after PM was 13.3 (± 9.6), 7.0 (± 3.3), 6.9 (± 2.8) and 7.4 (± 2.8) mm following MRT, US, and CE performed by a plastic surgeon and a general surgeon, respectively. The mean difference in estimated skin flap thickness was significant between MRT and the other measuring methods, while there was no significant difference between US and CE, nor between CE performed by the surgeons. The mean skin flap thickness was significantly affected by the age at PM. Following PM, necrosis was detected in 7/23 (30.4%) of the breasts in skin flaps ≤ 5 mm and in 5/46 (10.9%) of the breasts in skin flaps > 5 mm (OR 6.29; CI 1.20-32.94; p = 0.03)., Conclusion: The odds of getting postoperative necrosis was > 6 times higher in skin flaps ≤ 5 mm. Thus, if the degree of remaining glandular tissue is acceptably low, it is desirable to create skin flaps thicker than 5 mm to prevent wound healing problems after the PM procedure.

The incidence of reproductive diapause is a critical aspect of life history in overwintering insects from temperate regions. Much has been learned about the timing, physiology and genetics of diapause in a range of insects, but how the multiple changes involved in this and other photoperiodically regulated traits are inter-related is not well understood. We performed quasinatural selection on reproduction under short photoperiods in a northern fly species, Drosophila montana , to trace the effects of photoperiodic selection on traits regulated by the photoperiodic timer and/or by a circadian clock system. Selection changed several traits associated with reproductive diapause, including the critical day length for diapause (CDL), the frequency of diapausing females under photoperiods that deviate from daily 24 h cycles and cold tolerance, towards the phenotypes typical of lower latitudes. However, selection had no effect on the period of free-running locomotor activity rhythm regulated by the circadian clock in fly brain. At a genomic level, selection induced extensive divergence from the control line in 16 gene clusters involved in signal transduction, membrane properties, immunologlobulins and development. These changes resembled those detected between latitudinally divergent D. montana populations in the wild and involved SNP divergence associated with several genes linked with diapause induction. Overall, our study shows that photoperiodic selection for reproduction under short photoperiods affects diapause-associated traits without disrupting the central clock network generating circadian rhythms in fly locomotor activity., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The ability to discriminate between diverse types of sensation is mediated by heterogeneous populations of peripheral sensory neurons. Human peripheral sensory neurons are inaccessible for research and efforts to study their development and disease have been hampered by the availability of relevant model systems. The in vitro differentiation of peripheral sensory neurons from human embryonic stem cells therefore provides an attractive alternative since an unlimited source of biological material can be generated for studies that specifically address development and injury. The work presented in this study describes the derivation of peripheral sensory neurons from human embryonic stem cells using small molecule inhibitors. The differentiated neurons express canonical- and modality-specific peripheral sensory neuron markers with subsets exhibiting functional properties of human nociceptive neurons that include tetrodotoxin-resistant sodium currents and repetitive action potentials. Moreover, the derived cells associate with human donor Schwann cells and can be used as a model system to investigate the molecular mechanisms underlying neuronal death following peripheral nerve injury. The quick and efficient derivation of genetically diverse peripheral sensory neurons from human embryonic stem cells offers unlimited access to these specialised cell types and provides an invaluable in vitro model system for future studies.

Peripheral nerve injuries induce significant sensory neuronal cell death in the dorsal root ganglia (DRG); however, the role of specific apoptotic pathways is still unclear. In this study, we performed peripheral nerve transection on adult rats, after which the corresponding DRGs were harvested at 7, 14, and 28 days after injury for subsequent molecular analyses with quantitative reverse transcription-PCR, western blotting, and immunohistochemistry. Nerve injury led to increased levels of caspase-3 mRNA and active caspase-3 protein in the DRG. Increased expression of caspase-8, caspase-12, caspase-7, and calpain suggested that both the extrinsic and the endoplasmic reticulum (ER) stress-mediated apoptotic pathways were activated. Phosphorylation of protein kinase R-like ER kinase further implied the involvement of ER-stress in the DRG. Phosphorylated protein kinase R-like ER kinase was most commonly associated with isolectin B4 (IB4)-positive neurons in the DRG and this may provide an explanation for the increased susceptibility of these neurons to die following nerve injury, likely in part because of an activation of the ER-stress response.

Introduction: Previously we showed that a fibrin glue conduit with human mesenchymal stem cells (hMSCs) and cyclosporine A (CsA) enhanced early nerve regeneration. In this study long term effects of this conduit are investigated., Methods: In a rat model, the sciatic nerve was repaired with fibrin conduit containing fibrin matrix, fibrin conduit containing fibrin matrix with CsA treatment and fibrin conduit containing fibrin matrix with hMSCs and CsA treatment, and also with nerve graft as control., Results: At 12 weeks 34% of motoneurons of the control group regenerated axons through the fibrin conduit. CsA treatment alone or with hMSCs resulted in axon regeneration of 67% and 64% motoneurons respectively. The gastrocnemius muscle weight was reduced in the conduit with fibrin matrix. The treatment with CsA or CsA with hMSCs induced recovery of the muscle weight and size of fast type fibers towards the levels of the nerve graft group., Discussion: The transplantation of hMSCs for peripheral nerve injury should be optimized to demonstrate their beneficial effects. The CsA may have its own effect on nerve regeneration., Competing Interests: Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2018.)

Retrograde cell death in sensory dorsal root ganglion cells following peripheral nerve injury is well established. However, available data regarding the underlying mechanism behind injury induced motoneuron death are conflicting. By comparing morphological and molecular changes in spinal motoneurons after L4-L5 ventral root avulsion (VRA) and distal peripheral nerve axotomy (PNA) 7 and 14 days postoperatively, we aimed to gain more insight about the mechanism behind injury-induced motoneuron degeneration. Morphological changes in spinal cord were assessed by using quantitative immunohistochemistry. Neuronal degeneration was revealed by decreased immunostaining for microtubule-associated protein-2 in dendrites and synaptophysin in presynaptic boutons after both VRA and PNA. Significant motoneuron atrophy was already observed at 7 days post-injury, independently of injury type. Immunostaining for ED1 reactive microglia was significantly elevated in all experimental groups, as well as the astroglial marker glial fibrillary acidic protein (GFAP). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of the ventral horn from L4-L5 spinal cord segments revealed a significant upregulation of genes involved in programmed cell death including caspase-3, caspase-8, and related death receptors TRAIL-R, tumor necrosis factor (TNF)-R, and Fas following VRA. In contrast, following PNA, caspase-3 and the death receptor gene expression levels did not differ from the control, and there was only a modest increased expression of caspase-8. Moreover, the altered gene expression correlated with protein changes. These results show that the spinal motoneurons reacted in a similar fashion with respect to morphological changes after both proximal and distal injury. However, the increased expression of caspase-3, caspase-8, and related death receptors after VRA suggest that injury- induced motoneuron degeneration is mediated through an apoptotic mechanism, which might involve both the intrinsic and the extrinsic pathways.

Despite surgical innovation, the sensory and motor outcome after a peripheral nerve injury remains incomplete. One contributing factor to the poor outcome is prolonged denervation of the target organ, leading to apoptosis of both mature myofibres and satellite cells with subsequent replacement of the muscle tissue with fibrotic scar and adipose tissue. In this study, we investigated the expression of myogenic transcription factors, muscle specific microRNAs and muscle-specific E3 ubiquitin ligases at several time points following denervation in two different muscles, the gastrocnemius (containing predominantly fast type fibres) and soleus (slow type) muscles, since these molecules may influence the degree of atrophy following denervation. Both muscles exhibited significant atrophy (compared with the contra-lateral sides) at 7 days following either a nerve transection or crush injury. In the crush model, the soleus muscle showed significantly increased muscle weights at days 14 and 28 which was not the case for the gastrocnemius muscle which continued to atrophy. There was a significantly more pronounced up-regulation of MyoD expression in the denervated soleus muscle compared with the gastrocnemius muscle. Conversely, myogenin was more markedly elevated in the gastrocnemius versus soleus muscles. The muscles also showed significantly contrasting transcriptional regulation of the microRNAs miR-1 and miR-206. MuRF1 and Atrogin-1 showed the highest levels of expression in the denervated gastrocnemius muscle. This study provides further insights regarding the intracellular regulatory molecules that generate and maintain distinct patterns of gene expression in different fibre types following peripheral nerve injury.

Long noncoding RNAs (lncRNAs) are one of the most intensively studied groups of noncoding elements. Debate continues over what proportion of lncRNAs are functional or merely represent transcriptional noise. Although characterization of individual lncRNAs has identified approximately 200 functional loci across the Eukarya, general surveys have found only modest or no evidence of long-term evolutionary conservation. Although this lack of conservation suggests that most lncRNAs are nonfunctional, the possibility remains that some represent recent evolutionary innovations. We examine recent selection pressures acting on lncRNAs in mouse populations. We compare patterns of within-species nucleotide variation at approximately 10,000 lncRNA loci in a cohort of the wild house mouse, Mus musculus castaneus, with between-species nucleotide divergence from the rat (Rattus norvegicus). Loci under selective constraint are expected to show reduced nucleotide diversity and divergence. We find limited evidence of sequence conservation compared with putatively neutrally evolving ancestral repeats (ARs). Comparisons of sequence diversity and divergence between ARs, protein-coding (PC) exons and lncRNAs, and the associated flanking regions, show weak, but significantly lower levels of sequence diversity and divergence at lncRNAs compared with ARs. lncRNAs conserved deep in the vertebrate phylogeny show lower within-species sequence diversity than lncRNAs in general. A set of 74 functionally characterized lncRNAs show levels of diversity and divergence comparable to PC exons, suggesting that these lncRNAs are under substantial selective constraints. Our results suggest that, in mouse populations, most lncRNA loci evolve at rates similar to ARs, whereas older lncRNAs tend to show signals of selection similar to PC genes., (© The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Despite advances in surgical techniques for peripheral nerve repair, functional restitution remains incomplete. The timing of surgery is one factor influencing the extent of recovery but it is not yet clearly defined how long a delay may be tolerated before repair becomes futile. In this study, rats underwent sciatic nerve transection before immediate (0) or 1, 3, or 6 months delayed repair with a nerve graft. Regeneration of spinal motoneurons, 13 weeks after nerve repair, was assessed using retrograde labeling. Nerve tissue was also collected from the proximal and distal stumps and from the nerve graft, together with the medial gastrocnemius (MG) muscles. A dramatic decline in the number of regenerating motoneurons and myelinated axons in the distal nerve stump was observed in the 3- and 6-months delayed groups. After 3 months delay, the axonal number in the proximal stump increased 2-3 folds, accompanied by a smaller axonal area. RT-PCR of distal nerve segments revealed a decline in Schwann cells (SC) markers, most notably in the 3 and 6 month delayed repair samples. There was also a progressive increase in fibrosis and proteoglycan scar markers in the distal nerve with increased delayed repair time. The yield of SC isolated from the distal nerve segments progressively fell with increased delay in repair time but cultured SC from all groups proliferated at similar rates. MG muscle at 3- and 6-months delay repair showed a significant decline in weight (61% and 27% compared with contra-lateral side). Muscle fiber atrophy and changes to neuromuscular junctions were observed with increased delayed repair time suggestive of progressively impaired reinnervation. This study demonstrates that one of the main limiting factors for nerve regeneration after delayed repair is the distal stump. The critical time point after which the outcome of regeneration becomes too poor appears to be 3-months.

To assess the role of smoking and heterozygous (PiMZ) alpha 1-antitrypsin deficiency as risk factors in the pathogenesis of emphysema, we compared results of FEV1.0 (and FEV%) measurements in a random population sample of 56-year-old men with those obtained in an investigation 6 years earlier. We studied 32 PiMZ heterozygotes (14 smokers) and 31 PiM controls (13 smokers), representing 81% of the initial series. The annual decline in FEV1.0 values in non-smoking 56-year-old PiMZ men did not differ from PiM controls (smokers or non-smokers). In contrast, smoking heterozygotes showed a significantly higher mean annual decrease in FEV1.0 than non-smoking heterozygotes (75 ml and 40 ml/year respectively). In spite of this evidence of a modest accelerating effect on lung ageing among smoking PiMZ subjects during the 6 years covered by the study, no increased prevalence of clinical obstructive lung disease was noted.