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BACKGROUNDPreclinical studies suggest that cholesterol accumulation leads to insulin resistance. We previously reported that alterations in a monocyte cholesterol metabolism transcriptional network (CMTN) - suggestive of cellular cholesterol accumulation - were cross-sectionally associated with obesity and type 2 diabetes (T2D). Here, we sought to determine whether the CMTN alterations independently predict incident prediabetes/T2D risk, and correlate with cellular cholesterol accumulation.METHODSMonocyte mRNA expression of 11 CMTN genes was quantified among 934 Multi-Ethnic Study of Atherosclerosis (MESA) participants free of prediabetes/T2D; cellular cholesterol was measured in a subset of 24 monocyte samples.RESULTSDuring a median 6-year follow-up, lower expression of 3 highly correlated LXR target genes - ABCG1 and ABCA1 (cholesterol efflux) and MYLIP (cholesterol uptake suppression) - and not other CMTN genes, was significantly associated with higher risk of incident prediabetes/T2D. Lower expression of the LXR target genes correlated with higher cellular cholesterol levels (e.g., 47% of variance in cellular total cholesterol explained by ABCG1 expression). Further, adding the LXR target genes to overweight/obesity and other known predictors significantly improved prediction of incident prediabetes/T2D.CONCLUSIONThese data suggest that the aberrant LXR/ABCG1-ABCA1-MYLIP pathway (LAAMP) is a major T2D risk factor and support a potential role for aberrant LAAMP and cellular cholesterol accumulation in diabetogenesis.FUNDINGThe MESA Epigenomics and Transcriptomics Studies were funded by NIH grants 1R01HL101250, 1RF1AG054474, R01HL126477, R01DK101921, and R01HL135009. This work was supported by funding from NIDDK R01DK103531 and NHLBI R01HL119962.

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Globe-LFMC 2.0, an updated version of Globe-LFMC, is a comprehensive dataset of over 280,000 Live Fuel Moisture Content (LFMC) measurements. These measurements were gathered through field campaigns conducted in 15 countries spanning 47 years. In contrast to its prior version, Globe-LFMC 2.0 incorporates over 120,000 additional data entries, introduces more than 800 new sampling sites, and comprises LFMC values obtained from samples collected until the calendar year 2023. Each entry within the dataset provides essential information, including date, geographical coordinates, plant species, functional type, and, where available, topographical details. Moreover, the dataset encompasses insights into the sampling and weighing procedures, as well as information about land cover type and meteorological conditions at the time and location of each sampling event. Globe-LFMC 2.0 can facilitate advanced LFMC research, supporting studies on wildfire behaviour, physiological traits, ecological dynamics, and land surface modelling, whether remote sensing-based or otherwise. This dataset represents a valuable resource for researchers exploring the diverse LFMC aspects, contributing to the broader field of environmental and ecological research.

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WLSplot is an R package used to easily analyze lifespan survival data, and display results graphically as a survival curve with useful labels and statistical information auto-generated from the data and added to the graph, within a single function. It is designed primarily with Caenorhabditis elegans lifespan data in mind initially but can easily be used for other types of survival data. The WLSplot GitHub repository provides a blank template spreadsheet to be used for collecting lifespan data, instructions on how to install and run WLSplot, and examples covering RNAi, Genotype, or Drug lifespan experimental set-ups. WLSplot can analyze and plot multiple experiments in bulk while correctly italicizing worm gene names and adding asterisks and p-values to the plot legend when a significantly different lifespan from the designated control lifespan is seen. This is returned as an editable scalable vector graphics (svg) file for each output, and WLSplot can also return the summary of the directly plotted data so that the researcher can do their own further manipulation, in addition to being able to edit the output svg files.

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The Military Health System directly supports the National Security and Defense Strategy priorities of modernizing capabilities, enhancing lethality, supporting alliances, building partnerships, and implementing reform. Trauma medicine training programs with partner nations is a key lever that can be pulled, using a risk-based decision-making process, to scale up efforts toward these national priorities.

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Sirt6 is a multifunctional enzyme that regulates diverse cellular processes such as metabolism, DNA repair, and aging. Overexpressing Sirt6 extends lifespan in mice, but the underlying cellular mechanisms are unclear. Drosophila melanogaster are an excellent model to study genetic regulation of lifespan; however, despite extensive study in mammals, very little is known about Sirt6 function in flies. Here, we characterized the Drosophila ortholog of Sirt6, dSirt6, and examined its role in regulating longevity; dSirt6 is a nuclear and chromatin-associated protein with NAD+-dependent histone deacetylase activity. dSirt6 overexpression (OE) in flies produces robust lifespan extension in both sexes, while reducing dSirt6 levels shortens lifespan. dSirt6 OE flies have normal food consumption and fertility but increased resistance to oxidative stress and reduced protein synthesis rates. Transcriptomic analyses reveal that dSirt6 OE reduces expression of genes involved in ribosome biogenesis, including many dMyc target genes. dSirt6 OE partially rescues many effects of dMyc OE, including increased nuclear size, up-regulation of ribosome biogenesis genes, and lifespan shortening. Last, dMyc haploinsufficiency does not convey additional lifespan extension to dSirt6 OE flies, suggesting dSirt6 OE is upstream of dMyc in regulating lifespan. Our results provide insight into the mechanisms by which Sirt6 OE leads to longer lifespan.

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The genomes of virtually all organisms contain repetitive sequences that are generated by the activity of transposable elements (transposons). Transposons are mobile genetic elements that can move from one genomic location to another; in this process, they amplify and increase their presence in genomes, sometimes to very high copy numbers. In this Review we discuss new evidence and ideas that the activity of retrotransposons, a major subgroup of transposons overall, influences and even promotes the process of ageing and age-related diseases in complex metazoan organisms, including humans. Retrotransposons have been coevolving with their host genomes since the dawn of life. This relationship has been largely competitive, and transposons have earned epithets such as 'junk DNA' and 'molecular parasites'. Much of our knowledge of the evolution of retrotransposons reflects their activity in the germline and is evident from genome sequence data. Recent research has provided a wealth of information on the activity of retrotransposons in somatic tissues during an individual lifespan, the molecular mechanisms that underlie this activity, and the manner in which these processes intersect with our own physiology, health and well-being.

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Our intent in presenting this information is to increase the awareness of the Special Operations Forces (SOF) medical community and the overall international medical/military communities about the North Atlantic Treaty Organization (NATO) military medicine's premiere Vigorous Warrior Exercises organized by NATO Centre of Excellence for Military Medicine (MILMED COE). The Vigorous Warrior medical exercise series is conducted biennially, with four successful iterations since 2011. These international medical exercises engage military medical elements that enhance NATO capabilities and ensure that new NATO medical concepts are being exercised and tested across the full capability-requirement spectrum. The primary aims of these exercises are to provide NATO and partner nations a multipurpose platform to collectively train their medical forces and personnel; test and experiment new concepts and doctrines; medically evaluate national or multinational medical treatment facilities in accordance with NATO doctrine; produce medical lessons identified and lessons learned; and provide the participants with multinational experience to enhance the provision of health care in NATO operations. These exercises directly strengthen partnerships, improve military medical interoperability, and demonstrate the Alliance's commitment to improving international military collaboration. More than 1,000 medical personnel from 26 NATO and partner nations successfully conducted the joint, multilevel, multinational, medical live exercise Vigorous Warrior 2017 (VW17) throughout three locations in Germany during 4-22 September 2017. This article details the highly successful VW17 and paves the way for a very bright future for the Alliance's military medicine as well as a Vigorous Warrior 2019.

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BACKGROUND: CD4+ T cells can be broadly divided into naïve and memory subsets, each of which are differentially impaired by the aging process. It is unclear if and how these differences are reflected at the transcriptomic level. We performed microarray profiling on RNA derived from naïve (CD44low) and memory (CD44high) CD4+ T cells derived from young (2-3 month) and old (28 month) mice, in order to better understand the mechanisms of age-related functional alterations in both subsets. We also performed follow-up bioinformatic analyses in order to determine the functional consequences of gene expression changes in both of these subsets, and identify regulatory factors potentially responsible for these changes. RESULTS: We found 185 and 328 genes differentially expressed (FDR ≤ 0.05) in young vs. old naïve and memory cells, respectively, with 50 genes differentially expressed in both subsets. Functional annotation analyses highlighted an increase in genes involved in apoptosis specific to aged naïve cells. Both subsets shared age-related increases in inflammatory signaling genes, along with a decrease in oxidative phosphorylation genes. Cis-regulatory analyses revealed enrichment of multiple transcription factor binding sites near genes with age-associated expression, in particular NF-κB and several forkhead box transcription factors. Enhancer associated histone modifications were enriched near genes down-regulated in naïve cells. Comparison of our results with previous mouse and human datasets indicates few overlapping genes overall, but suggest consistent up-regulation of Casp1 and Il1r2, and down-regulation of Foxp1 in both mouse and human CD4+ T cells. CONCLUSIONS: The transcriptomes of naïve and memory CD4+ T cells are distinctly affected by the aging process. However, both subsets exhibit a common increase inflammatory genes and decrease in oxidative phosphorylation genes. NF-κB, forkhead box, and Myc transcription factors are implicated as upstream regulators of these gene expression changes in both subsets, with enhancer histone modifications potentially driving unique changes unique to naïve cells. Finally we conclude that there is little overlap in age-related gene expression changes between humans and mice; however, age-related alterations in a small subset of genes may be conserved.

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The goal of magnetic field-assisted gene transfer is to enhance internalization of exogenous nucleic acids by association with magnetic nanoparticles (MNPs). This technique named magnetofection is particularly useful in difficult-to-transfect cells. It is well known that human, mouse, and rat skeletal muscle cells suffer a maturation-dependent loss of susceptibility to Recombinant Adenoviral vector (RAd) uptake. In postnatal, fully differentiated myofibers, the expression of the primary Coxsackie and Adenoviral membrane receptor (CAR) is severely downregulated representing a main hurdle for the use of these vectors in gene transfer/therapy. Here we demonstrate that assembling of Recombinant Adenoviral vectors with suitable iron oxide MNPs into magneto-adenovectors (RAd-MNP) and further exposure to a gradient magnetic field enables to efficiently overcome transduction resistance in skeletal muscle cells. Expression of Green Fluorescent Protein and Insulin-like Growth Factor 1 was significantly enhanced after magnetofection with RAd-MNPs complexes in C2C12 myotubes in vitro and mouse skeletal muscle in vivo when compared to transduction with naked virus. These results provide evidence that magnetofection, mainly due to its membrane-receptor independent mechanism, constitutes a simple and effective alternative to current methods for gene transfer into traditionally hard-to-transfect biological models.

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BACKGROUND: Tobacco smoke contains numerous agonists of the aryl hydrocarbon receptor (AhR) pathway, and activation of the AhR pathway was shown to promote atherosclerosis in mice. Intriguingly, cigarette smoking is most strongly and robustly associated with DNA modifications to an AhR pathway gene, the AhR repressor (AHRR). We hypothesized that altered AHRR methylation in monocytes, a cell type sensitive to cigarette smoking and involved in atherogenesis, may be a part of the biological link between cigarette smoking and atherosclerosis. METHODS AND RESULTS: DNA methylation profiles of AHRR in monocytes (542 CpG sites ± 150 kb of AHRR, using Illumina 450K array) were integrated with smoking habits and ultrasound-measured carotid plaque scores from 1256 participants of the Multi-Ethnic Study of Atherosclerosis (MESA). Methylation of cg05575921 significantly associated (P=6.1 × 10(-134)) with smoking status (current versus never). Novel associations between cg05575921 methylation and carotid plaque scores (P=3.1 × 10(-10)) were identified, which remained significant in current and former smokers even after adjusting for self-reported smoking habits, urinary cotinine, and well-known cardiovascular disease risk factors. This association replicated in an independent cohort using hepatic DNA (n=141). Functionally, cg05575921 was located in a predicted gene expression regulatory element (enhancer) and had methylation correlated with AHRR mRNA profiles (P=1.4 × 10(-17)) obtained from RNA sequencing conducted on a subset (n=373) of the samples. CONCLUSIONS: These findings suggest that AHRR methylation may be functionally related to AHRR expression in monocytes and represents a potential biomarker of subclinical atherosclerosis in smokers.

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The voltage-gated calcium channel (Cav) ß1a subunit (Cavß1a) plays an important role in excitation-contraction coupling (ECC), a process in the myoplasm that leads to muscle-force generation. Recently, we discovered that the Cavß1a subunit travels to the nucleus of skeletal muscle cells where it helps to regulate gene transcription. To determine how it travels to the nucleus, we performed a yeast two-hybrid screening of the mouse fast skeletal muscle cDNA library and identified an interaction with troponin T3 (TnT3), which we subsequently confirmed by co-immunoprecipitation and co-localization assays in mouse skeletal muscle in vivo and in cultured C2C12 muscle cells. Interacting domains were mapped to the leucine zipper domain in TnT3 COOH-terminus (160-244 aa) and Cavß1a NH2-terminus (1-99 aa), respectively. The double fluorescence assay in C2C12 cells co-expressing TnT3/DsRed and Cavß1a/YFP shows that TnT3 facilitates Cavß1a nuclear recruitment, suggesting that the two proteins play a heretofore unknown role during early muscle differentiation in addition to their classical role in ECC regulation.

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BACKGROUND: Transcriptomic studies hold great potential towards understanding the human aging process. Previous transcriptomic studies have identified many genes with age-associated expression levels; however, small samples sizes and mixed cell types often make these results difficult to interpret. RESULTS: Using transcriptomic profiles in CD14+ monocytes from 1,264 participants of the Multi-Ethnic Study of Atherosclerosis (aged 55-94 years), we identified 2,704 genes differentially expressed with chronological age (false discovery rate, FDR ≤ 0.001). We further identified six networks of co-expressed genes that included prominent genes from three pathways: protein synthesis (particularly mitochondrial ribosomal genes), oxidative phosphorylation, and autophagy, with expression patterns suggesting these pathways decline with age. Expression of several chromatin remodeler and transcriptional modifier genes strongly correlated with expression of oxidative phosphorylation and ribosomal protein synthesis genes. 17% of genes with age-associated expression harbored CpG sites whose degree of methylation significantly mediated the relationship between age and gene expression (p < 0.05). Lastly, 15 genes with age-associated expression were also associated (FDR ≤ 0.01) with pulse pressure independent of chronological age. Comparing transcriptomic profiles of CD14+ monocytes to CD4+ T cells from a subset (n = 423) of the population, we identified 30 age-associated (FDR < 0.01) genes in common, while larger sets of differentially expressed genes were unique to either T cells (188 genes) or monocytes (383 genes). At the pathway level, a decline in ribosomal protein synthesis machinery gene expression with age was detectable in both cell types. CONCLUSIONS: An overall decline in expression of ribosomal protein synthesis genes with age was detected in CD14+ monocytes and CD4+ T cells, demonstrating that some patterns of aging are likely shared between different cell types. Our findings also support cell-specific effects of age on gene expression, illustrating the importance of using purified cell samples for future transcriptomic studies. Longitudinal work is required to establish the relationship between identified age-associated genes/pathways and aging-related diseases.

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Age-related variations in DNA methylation have been reported; however, the functional relevance of these differentially methylated sites (age-dMS) are unclear. Here we report potentially functional age-dMS, defined as age- and cis-gene expression-associated methylation sites (age-eMS), identified by integrating genome-wide CpG methylation and gene expression profiles collected ex vivo from circulating T cells (227 CD4+ samples) and monocytes (1,264 CD14+ samples, age range: 55-94 years). None of the age-eMS detected in 227 T-cell samples are detectable in 1,264 monocyte samples, in contrast to the majority of age-dMS detected in T cells that replicated in monocytes. Age-eMS tend to be hypomethylated with older age, located in predicted enhancers and preferentially linked to expression of antigen processing and presentation genes. These results identify and characterize potentially functional age-related methylation in human T cells and monocytes, and provide novel insights into the role age-dMS may have in the aging process.

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Voltage-gated calcium channel (Cav) ß subunits are auxiliary subunits to Cavs. Recent reports show Cavß subunits may enter the nucleus and suggest a role in transcriptional regulation, but the physiological relevance of this localization remains unclear. We sought to define the nuclear function of Cavß in muscle progenitor cells (MPCs). We found that Cavß1a is expressed in proliferating MPCs, before expression of the calcium conducting subunit Cav1.1, and enters the nucleus. Loss of Cavß1a expression impaired MPC expansion in vitro and in vivo and caused widespread changes in global gene expression, including up-regulation of myogenin. Additionally, we found that Cavß1a localizes to the promoter region of a number of genes, preferentially at noncanonical (NC) E-box sites. Cavß1a binds to a region of the Myog promoter containing an NC E-box, suggesting a mechanism for inhibition of myogenin gene expression. This work indicates that Cavß1a acts as a Cav-independent regulator of gene expression in MPCs, and is required for their normal expansion during myogenic development.

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Troponin T (TnT) is known to mediate the interaction between Tn complex and tropomyosin (Tm), which is essential for calcium-activated striated muscle contraction. This regulatory function takes place in the myoplasm, where TnT binds Tm. However, recent findings of troponin I and Tm nuclear translocation in Drosophila and mammalian cells imply other roles for the Tn-Tm complex. We hypothesized that TnT plays a nonclassical role through nuclear translocation. Immunoblotting with different antibodies targeting the NH2- or COOH-terminal region uncovered a pool of fast skeletal muscle TnT3 localized in the nuclear fraction of mouse skeletal muscle as either an intact or fragmented protein. Construction of TnT3-DsRed fusion proteins led to the further observation that TnT3 fragments are closely related to nucleolus and RNA polymerase activity, suggesting a role for TnT3 in regulating transcription. Functionally, overexpression of TnT3 fragments produced significant defects in nuclear shape and caused high levels of apoptosis. Interestingly, nuclear TnT3 and its fragments were highly regulated by aging, thus creating a possible link between the deleterious effects of TnT3 and sarcopenia. We propose that changes in nuclear TnT3 and its fragments cause the number of myonuclei to decrease with age, contributing to muscle damage and wasting.

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Escape from cellular senescence induction is a potent mechanism for chemoresistance. Cellular senescence can be induced in breast cancer cell lines by the removal of estrogen signaling with tamoxifen or by the accumulation of DNA damage induced by the chemotherapeutic drug doxorubicin. Long term culturing of the hormone-sensitive breast cancer cell line MCF-7 in doxorubicin (MCF-7/DoxR) reduced the ability of doxorubicin, but not tamoxifen, to induce senescence. Two pathways that are often upregulated in chemo- and hormonal-resistance are the PI3K/PTEN/Akt/mTOR and Ras/Raf/MEK/ERK pathways. To determine if active Akt-1 and Raf-1 can influence drug-induced senescence, we stably introduced activated ΔAkt-1(CA) and ΔRaf-1(CA) into drug-sensitive and doxorubicin-resistant cells. Expression of a constitutively-active Raf-1 construct resulted in higher baseline senescence, indicating these cells possessed the ability to undergo oncogene-induced-senescence. Constitutive activation of the Akt pathway significantly decreased drug-induced senescence in response to doxorubicin but not tamoxifen in MCF-7 cells. However, constitutive Akt-1 activation in drug-resistant cells containing high levels of active ERK completely escaped cellular senescence induced by doxorubicin and tamoxifen. These results indicate that up regulation of the Ras/PI3K/PTEN/Akt/mTOR pathway in the presence of elevated Ras/Raf/MEK/ERK signaling together can contribute to drug-resistance by diminishing cell senescence in response to chemotherapy. Understanding how breast cancers containing certain oncogenic mutations escape cell senescence in response to chemotherapy and hormonal based therapies may provide insights into the design of more effective drug combinations for the treatment of breast cancer.

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Ca2+ release from the sarcoplasmic reticulum (SR) into the cytosol is a crucial part of excitation-contraction (E-C) coupling. Excitation-contraction uncoupling, a deficit in Ca2+ release from the SR, is thought to be responsible for at least some of the loss in specific force observed in aging skeletal muscle. Excitation-contraction uncoupling may be caused by alterations in expression of the voltage-dependent calcium channel alpha1s (CaV1.1) and beta1a (CaVbeta1a) subunits, both of which are necessary for E-C coupling to occur. While previous studies have found CaV1.1 expression declines in old rodents, CaVbeta1a expression has not been previously examined in aging models. Western blot analysis shows a substantial increase of CaVbeta1a expression over the full lifespan of Friend Virus B (FVB) mice. To examine the specific effects of CaVbeta1a overexpression, a CaVbeta1a-YFP plasmid was electroporated in vivo into young animals. The resulting increase in expression of CaVbeta1a corresponded to decline of CaV1.1 over the same time period. YFP fluorescence, used as a measure of CaVbeta1a-YFP expression in individual fibers, also showed an inverse relationship with charge movement, measured using the whole-cell patch-clamp technique. Specific force was significantly reduced in young CaVbeta1a-YFP electroporated muscle fibers compared with sham-electroporated, age-matched controls. siRNA interference of CaVbeta1a in young muscles reduced charge movement, while charge movement in old was restored to young control levels. These studies imply CaVbeta1a serves as both a positive and negative regulator CaV1.1 expression, and that endogenous overexpression of CaVbeta1a during old age may play a role in the loss of specific force.

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