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Abstract Background Transfection agents comprised of cationic lipid preparations are widely used to transfect cell lines in culture with specific recombinant complementary DNA molecules. We have found that cells in culture are often resistant to stimulation with insulin subsequent to treatment with transfection agents such as LipofectAMINE 2000™ and FuGENE-6™. This is seen with a variety of different readouts, including insulin receptor signalling, glucose uptake into muscle cells, phosphorylation of protein kinase B and reporter gene activity in a variety of different cell types Results We now show that this is due in part to the fact that cationic lipid agents activate the insulin receptor fully during typical transfection experiments, which is then down-regulated. In attempts to circumvent this problem, we investigated the effects of increasing concentrations of LipofectAMINE 2000™ on insulin receptor phosphorylation in Chinese hamster ovary cells expressing the human insulin receptor. In addition, the efficiency of transfection that is supported by the same concentrations of transfection reagent was studied by using a green fluorescent protein construct. Our data indicate that considerably lower concentrations of LipofectAMINE 2000™ can be used than are recommended by the manufacturers. This is without sacrificing transfection efficiency markedly and avoids the problem of reducing insulin receptor expression in the cells. Conclusion Widely-used cationic lipid transfection reagents cause a state of insulin unresponsiveness in cells in culture due to fully activating and subsequently reducing the expression of the receptor in cells. This phenomenon can be avoided by reducing the concentration of reagent used in the transfection process.

In cell biology, ribosomal RNA (rRNA) 2' O -methyl (2'- O -Me) is the most prevalent posttranscriptional chemical modification contributing to ribosome heterogeneity. The modification involves a family of small nucleolar RNAs (snoRNAs) and is specified by box C/D snoRNAs (SNORDs). Given the importance of ribosome biogenesis for skeletal muscle growth, we asked if rRNA 2'- O -Me in nascent ribosomes synthesized in response to a growth stimulus is an unrecognized mode of ribosome heterogeneity in muscle. To determine the pattern and dynamics of 2'- O -Me rRNA, we used a sequencing-based profiling method called RiboMeth-seq (RMS). We applied this method to tissue-derived rRNA of skeletal muscle and rRNA specifically from the muscle fiber using an inducible myofiber-specific RiboTag mouse in sedentary and mechanically overloaded conditions. These analyses were complemented by myonuclear-specific small RNA sequencing to profile SNORDs and link the rRNA epitranscriptome to known regulatory elements generated within the muscle fiber. We demonstrate for the first time that mechanical overload of skeletal muscle 1 ) induces decreased 2'- O -Me at a subset of skeletal muscle rRNA and 2 ) alters the SNORD profile in isolated myonuclei. These findings point to a transient diversification of the ribosome pool via 2'- O -Me during growth and adaptation in skeletal muscle. These findings suggest changes in ribosome heterogeneity at the 2'- O -Me level during muscle hypertrophy and lay the foundation for studies investigating the functional implications of these newly identified "growth-induced" ribosomes. NEW & NOTEWORTHY Ribosomal RNAs (rRNAs) are posttranscriptionally modified by 2' O -methyl (2'- O -Me). This study applied RiboMeth-seq (RMS) to detect changes in 2'- O -Me levels during skeletal muscle hypertrophy, uncovering transient diversification of the ribosome pool in skeletal muscle fibers. This work implies a role for ribosome heterogeneity in skeletal muscle growth and adaptation.

Background: Polycystic ovary syndrome's (PCOS) main feature is hyperandrogenism, which is linked to a higher risk of metabolic disorders. Gene expression analyses in adipose tissue and skeletal muscle reveal dysregulated metabolic pathways in women with PCOS, but these differences do not necessarily lead to changes in protein levels and biological function., Methods: To advance our understanding of the molecular alterations in PCOS, we performed global proteomic and phosphorylation site analysis using tandem mass spectrometry, and analyzed gene expression and methylation. Adipose tissue and skeletal muscle were collected at baseline from 10 women with and without PCOS, and in women with PCOS after 5 weeks of treatment with electrical stimulation., Results: Perilipin-1, a protein that typically coats the surface of lipid droplets in adipocytes, was increased whereas proteins involved in muscle contraction and type I muscle fiber function were downregulated in PCOS muscle. Proteins in the thick and thin filaments had many altered phosphorylation sites, indicating differences in protein activity and function. A mouse model was used to corroborate that androgen exposure leads to a shift in muscle fiber type in controls but not in skeletal muscle-specific androgen receptor knockout mice. The upregulated proteins in muscle post treatment were enriched in pathways involved in extracellular matrix organization and wound healing, which may reflect a protective adaptation to repeated contractions and tissue damage due to needling. A similar, albeit less pronounced, upregulation in extracellular matrix organization pathways was also seen in adipose tissue., Conclusions: Our results suggest that hyperandrogenic women with PCOS have higher levels of extra-myocellular lipids and fewer oxidative insulin-sensitive type I muscle fibers. These could be key factors leading to insulin resistance in PCOS muscle while electric stimulation-induced tissue remodeling may be protective., Funding: Swedish Research Council (2020-02485, 2022-00550, 2020-01463), Novo Nordisk Foundation (NNF22OC0072904), and IngaBritt and Arne Lundberg Foundation. Clinical trial number NTC01457209., Competing Interests: ES, GE, MM, VR, HL, JB, MS, CP, BJ, VE, RB, EN, CL, JN, IW, NT, JL, AB No competing interests declared, (© 2023, Stener-Victorin et al.)

Climate warming has been observed as the main cause of changes in diversity, community composition, and spatial distribution of different plant and invertebrate species. Due to even stronger warming compared to the global mean, bumblebees in alpine ecosystems are particularly exposed to these changes. To investigate the effects of climate warming, we sampled bumblebees along an elevational gradient, compared the records with data from 1935 and 1936, and related our results to climate models. We found that bumblebee community composition differed significantly between sampling periods and that increasing temperatures in spring were the most plausible factor explaining these range shifts. In addition, species diversity estimates were significantly lower compared to historical records. The number of socio-parasitic species was significantly higher in the historical communities, while recent communities showed increases in climate generalists and forest species at lower elevations. Nevertheless, no significant changes in community-weighted means of a species temperature index (STI) or the number of cold-adapted species were detected, likely due to the historical data resolution. We conclude that the composition and functionality of bumblebee communities in the study area have been significantly affected by climate warming, with changes in land use and vegetation cover likely playing an additional important role.

Complex biotic networks of invaders and their new environments pose immense challenges for researchers aiming to predict current and future occupancy of introduced species. This might be especially true for invasive bees, as they enter novel trophic interactions. Little attention has been paid to solitary, invasive wild bees, despite their increasing recognition as a potential global threat to biodiversity. Here, we present the first comprehensive species distribution modelling approach targeting the invasive bee Megachile sculpturalis, which is currently undergoing parallel range expansion in North America and Europe. While the species has largely colonised the most highly suitable areas of North America over the past decades, its invasion of Europe seems to be in its early stages. We showed that its current distribution is largely explained by anthropogenic factors, suggesting that its spread is facilitated by road and maritime traffic, largely beyond its intrinsic dispersal ability. Our results suggest that M. sculpturalis is likely to be negatively affected by future climate change in North America, while in Europe the potential suitable areas at-risk of invasion remain equally large. Based on our study, we emphasise the role of expert knowledge for evaluation of ecologically meaningful variables implemented and interpreted for species distribution modelling. We strongly recommend that the monitoring of this and other invasive pollinator species should be prioritised in areas identified as at-risk, alongside development of effective management strategies., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Invasive species are increasingly driving biodiversity decline, and knowledge of colonization dynamics, including both drivers and dispersal modes, are important to prevent future invasions. The bee species Megachile sculpturalis (Hymenoptera: Megachilidae), native to East-Asia, was first recognized in Southeast-France in 2008, and has since spread throughout much of Europe. The spread is very fast, and colonization may result from multiple fronts., Result: To track the history of this invasion, codominant markers were genotyped using Illumina sequencing and the invasion history and degree of connectivity between populations across the European invasion axis were investigated. Distinctive genetic clusters were detected with east-west differentiations in Middle-Europe., Conclusion: We hypothesize that the observed cluster formation resulted from multiple, independent introductions of the species to the European continent. This study draws a first picture of an early invasion stage of this wild bee and forms a foundation for further investigations, including studies of the species in their native Asian range and in the invaded range in North America.

Biodiversity monitoring programs are the baseline of species abundancy studies, which in case of introduced species are especially critical. Megachile sculpturalis Smith, 1853 native to Eastern-Asia, constitutes the first ever recorded wild bee species accidently introduced in Europe. Since its first discovery in 2008, M. sculpturalis has been spreading across the continent. By initiating a citizen science monitoring program, we aimed to investigate the occurrence pattern of M. sculpturalis. Within only two years after starting the project, 111 new reports from Switzerland, Liechtenstein and Austria were recorded. Comparably to other European countries, the population progressed remarkably fast from year to year expanding its area geographically but also ecologically by increasing its altitudinal range. The distribution pattern indicates human assisted jump-dispersal travelling on the major traffic routes of central Europe., Competing Interests: The authors declare that they have no conflict of interest. The commercial affiliation of one of our co-authors, does not alter our adherence to PLOS ONE policies on sharing data and materials.

DNA barcoding constitutes a supplemental genetically based characterization tool for the identification of species. Traditionally, the barcodes are generated with a length of 650 bp using standardized Sanger sequencing, but with the introduction of high-throughput sequencing (HTS) methods new opportunities for sequencing are available. To use HTS for barcode collection and identification, the amplification of shorter fragments is preferred. Reference DNA midi-barcodes of wild bees were produced using the Illumina MiSeq as well as the Sanger method. Although DNA midi-barcodes derived from Illumina were comparatively shorter (418 bp), their sequences were coherent to the morphological assignment of species. The Illumina barcodes proved to be effective and dealt better with some general limitations of DNA barcoding.

Regular exercise has widespread health benefits. Fundamental to these beneficial effects is the ability of the heart to intermittently and substantially increase its performance without incurring damage, but the underlying homeostatic mechanisms are unclear. We identify the ROS-generating NADPH oxidase-4 (Nox4) as an essential regulator of exercise performance in mice. Myocardial Nox4 levels increase during acute exercise and trigger activation of the transcription factor Nrf2, with the induction of multiple endogenous antioxidants. Cardiomyocyte-specific Nox4-deficient (csNox4KO) mice display a loss of exercise-induced Nrf2 activation, cardiac oxidative stress and reduced exercise performance. Cardiomyocyte-specific Nrf2-deficient (csNrf2KO) mice exhibit similar compromised exercise capacity, with mitochondrial and cardiac dysfunction. Supplementation with an Nrf2 activator or a mitochondria-targeted antioxidant effectively restores cardiac performance and exercise capacity in csNox4KO and csNrf2KO mice respectively. The Nox4/Nrf2 axis therefore drives a hormetic response that is required for optimal cardiac mitochondrial and contractile function during physiological exercise., Competing Interests: MH, AH, AN, NC, AL, IS, SH, JL, KS, AS, MZ No competing interests declared, MM Has a financial interest in and is on the scientific advisory board of Antipodean Pharmaceuticals, Inc which is commercialising MitoQ., (© 2018, Hancock et al.)

The inorganic anion nitrate (NO3 (-)), which is naturally enriched in certain vegetables (e.g., spinach and beetroot), has emerged as a dietary component that can regulate diverse bodily functions, including blood pressure, mitochondrial efficiency, and skeletal muscle force. It is not known if dietary nitrate improves cardiac contractility. To test this, mice were supplemented for 1-2 weeks with sodium nitrate in the drinking water at a dose similar to a green diet. The hearts from nitrate-treated mice showed increased left ventricular pressure and peak rate of pressure development as measured with the Langendorff heart technique. Cardiomyocytes from hearts of nitrate-treated and control animals were incubated with the fluorescent indicator Fluo-3 to measure cytoplasmic free [Ca(2+)] and fractional shortening. Cardiomyocytes from nitrate-treated mice displayed increased fractional shortening, which was linked to larger Ca(2+) transients. Moreover, nitrate hearts displayed increased protein expression of the L-type Ca(2+) channel/dihydropyridine receptor and peak L-type Ca(2+) channel currents. The nitrate-treated hearts displayed increased concentration of cAMP but unchanged levels of cGMP compared with controls. These findings provide the first evidence that dietary nitrate can affect the expression of important Ca(2+) handling proteins in the heart, resulting in increased cardiomyocyte Ca(2+) signaling and improved left ventricular contractile function. Our observation shows that dietary nitrate impacts cardiac function and adds understanding to inorganic nitrate as a physiological modulator.

Background: Ca(2+) influx through CaV1.1 is not required for skeletal muscle excitation-contraction coupling, but whether Ca(2+) permeation through CaV1.1 during sustained muscle activity plays a functional role in mammalian skeletal muscle has not been assessed., Methods: We generated a mouse with a Ca(2+) binding and/or permeation defect in the voltage-dependent Ca(2+) channel, CaV1.1, and used Ca(2+) imaging, western blotting, immunohistochemistry, proximity ligation assays, SUnSET analysis of protein synthesis, and Ca(2+) imaging techniques to define pathways modulated by Ca(2+) binding and/or permeation of CaV1.1. We also assessed fiber type distributions, cross-sectional area, and force frequency and fatigue in isolated muscles., Results: Using mice with a pore mutation in CaV1.1 required for Ca(2+) binding and/or permeation (E1014K, EK), we demonstrate that CaV1.1 opening is coupled to CaMKII activation and refilling of sarcoplasmic reticulum Ca(2+) stores during sustained activity. Decreases in these Ca(2+)-dependent enzyme activities alter downstream signaling pathways (Ras/Erk/mTORC1) that lead to decreased muscle protein synthesis. The physiological consequences of the permeation and/or Ca(2+) binding defect in CaV1.1 are increased fatigue, decreased fiber size, and increased Type IIb fibers., Conclusions: While not essential for excitation-contraction coupling, Ca(2+) binding and/or permeation via the CaV1.1 pore plays an important modulatory role in muscle performance.

Background: Transfection agents comprised of cationic lipid preparations are widely used to transfect cell lines in culture with specific recombinant complementary DNA molecules. We have found that cells in culture are often resistant to stimulation with insulin subsequent to treatment with transfection agents such as LipofectAMINE 2000 and FuGENE-6. This is seen with a variety of different readouts, including insulin receptor signalling, glucose uptake into muscle cells, phosphorylation of protein kinase B and reporter gene activity in a variety of different cell types, Results: We now show that this is due in part to the fact that cationic lipid agents activate the insulin receptor fully during typical transfection experiments, which is then down-regulated. In attempts to circumvent this problem, we investigated the effects of increasing concentrations of LipofectAMINE 2000 on insulin receptor phosphorylation in Chinese hamster ovary cells expressing the human insulin receptor. In addition, the efficiency of transfection that is supported by the same concentrations of transfection reagent was studied by using a green fluorescent protein construct. Our data indicate that considerably lower concentrations of LipofectAMINE 2000 can be used than are recommended by the manufacturers. This is without sacrificing transfection efficiency markedly and avoids the problem of reducing insulin receptor expression in the cells., Conclusion: Widely-used cationic lipid transfection reagents cause a state of insulin unresponsiveness in cells in culture due to fully activating and subsequently reducing the expression of the receptor in cells. This phenomenon can be avoided by reducing the concentration of reagent used in the transfection process.