Translocatome: a novel resource for the analysis of protein translocation between cellular organelles.

Here we present Translocatome, the first dedicated database of human translocating proteins (URL: http://translocatome.linkgroup.hu). The core of the Translocatome database is the manually curated data set of 213 human translocating proteins listing the source of their experimental validation, several details of their translocation mechanism, their local compartmentalized interactome, as well as their involvement in signalling pathways and disease development. In addition, using the well-established and widely used gradient boosting machine learning tool, XGBoost, Translocatome provides translocation probability values for 13 066 human proteins identifying 1133 and 3268 high- and low-confidence translocating proteins, respectively. The database has user-friendly search options with a UniProt autocomplete quick search and advanced search for proteins filtered by their localization, UniProt identifiers, translocation likelihood or data complexity. Download options of search results, manually curated and predicted translocating protein sets are available on its website. The update of the database is helped by its manual curation framework and connection to the previously published ComPPI compartmentalized protein-protein interaction database (http://comppi.linkgroup.hu). As shown by the application examples of merlin (NF2) and tumor protein 63 (TP63) Translocatome allows a better comprehension of protein translocation as a systems biology phenomenon and can be used as a discovery-tool in the protein translocation field.

Significant association between tumor mutational burden and immune-related adverse events during immune checkpoint inhibition therapies.

More than 2000 immuno-oncology agents are being tested or are in use as a result of the cancer immunotherapy revolution. Manipulation of co-inhibitory receptors has achieved tumor eradication in a minority of patients, but widespread immune-related adverse events (irAEs) compromised tolerance to healthy self-tissues in the majority. We have proposed that a major mechanism of irAEs is similar to a graft-versus-malignancy effect of graft-versus-host disease. To verify our hypothesis, we retrieved post-marketing data of adverse events from the U.S. Food and Drug Administration Adverse Event Reporting System. A significant positive correlation was revealed in 7677 patients between the reporting odds ratio of irAEs during immune checkpoint inhibitor therapy and the corresponding tumor mutational burden across 19 cancer types. These results can be interpreted to mean that the ICI drugs unleashed T cells against "altered-self," self, and tumors resulting in better overall survival.

MiStImm: an agent-based simulation tool to study the self-nonself discrimination of the adaptive immune response.

BACKGROUND: There is an increasing need for complex computational models to perform in silico experiments as an adjunct to in vitro and in vivo experiments in immunology. We introduce Microscopic Stochastic Immune System Simulator (MiStImm), an agent-based simulation tool, that is designed to study the self-nonself discrimination of the adaptive immune system. MiStImm can simulate some components of the humoral adaptive immune response, including T cells, B cells, antibodies, danger signals, interleukins, self cells, foreign antigens, and the interactions among them. The simulation starts after conception and progresses step by step (in time) driven by random simulation events. We also have provided tools to visualize and analyze the output of the simulation program. RESULTS: As the first application of MiStImm, we simulated two different immune models, and then we compared performances of them in the mean of self-nonself discrimination. The first model is a so-called conventional immune model, and the second model is based on our earlier T-cell model, called "one-signal model", which is developed to resolve three important paradoxes of immunology. Our new T-cell model postulates that a dynamic steady state coupled system is formed through low-affinity complementary TCR-MHC interactions between T cells and host cells. The new model implies that a significant fraction of the naive polyclonal T cells is recruited into the first line of defense against an infection. Simulation experiments using MiStImm have shown that the computational realization of the new model shows real patterns. For example, the new model develops immune memory and it does not develop autoimmune reaction despite the hypothesized, enhanced TCR-MHC interaction between T cells and self cells. Simulations also demonstrated that our new model gives better results to overcome a critical primary infection answering the paradox "how can a tiny fraction of human genome effectively compete with a vastly larger pool of mutating pathogen DNA?" CONCLUSION: The outcomes of our in silico experiments, presented here, are supported by numerous clinical trial observations from the field of immunotherapy. We hope that our results will encourage investigations to make in vitro and in vivo experiments clarifying questions about self-nonself discrimination of the adaptive immune system. We also hope that MiStImm or some concept in it will be useful to other researchers who want to implement or compare other immune models.

Soda pans of the Pannonian steppe harbor unique bacterial communities adapted to multiple extreme conditions.

Soda pans of the Pannonian steppe are unique environments regarding their physical and chemical characteristics: shallowness, high turbidity, intermittent character, alkaline pH, polyhumic organic carbon concentration, hypertrophic condition, moderately high salinity, sodium and carbonate ion dominance. The pans are highly productive environments with picophytoplankton predominance. Little is known about the planktonic bacterial communities inhabiting these aquatic habitats; therefore, amplicon sequencing and shotgun metagenomics were applied to reveal their composition and functional properties. Results showed a taxonomically complex bacterial community which was distinct from other soda lakes regarding its composition, e.g. the dominance of class Alphaproteobacteria was observed within phylum Proteobacteria. The shotgun metagenomic analysis revealed several functional gene components related to the harsh and at the same time hypertrophic environmental conditions, e.g. proteins involved in stress response, transport and hydrolase systems targeting phytoplankton-derived organic matter. This is the first detailed report on the indigenous planktonic bacterial communities coping with the multiple extreme conditions present in the unique soda pans of the Pannonian steppe.

The "Giant Virus Finder" discovers an abundance of giant viruses in the Antarctic dry valleys.

Mimivirus was identified in 2003 from a biofilm of an industrial water-cooling tower in England. Later, numerous new giant viruses were found in oceans and freshwater habitats, some of them having 2,500 genes. We have demonstrated their likely presence in four soil samples taken from the Kutch Desert (Gujarat, India). Here we describe a bioinformatics work-flow, called the "Giant Virus Finder" that is capable of discovering the likely presence of the genomes of giant viruses in metagenomic shotgun-sequenced datasets. The new workflow is applied to numerous hot and cold desert soil samples as well as some tundra- and forest soils. We show that most of these samples contain giant viruses, especially in the Antarctic dry valleys. The results imply that giant viruses could be frequent not only in aqueous habitats, but in a wide spectrum of soils on our planet.

Giant viruses of the Kutch Desert.

The Kutch Desert (Great Rann of Kutch, Gujarat, India) is a unique ecosystem: in the larger part of the year it is a hot, salty desert that is flooded regularly in the Indian monsoon season. In the dry season, the crystallized salt deposits form the "white desert" in large regions. The first metagenomic analysis of the soil samples of Kutch was published in 2013, and the data were deposited in the NCBI Sequence Read Archive. At the same time, the sequences were analyzed phylogenetically for prokaryotes, especially for bacteria. In the present work, we identified DNA sequences of recently discovered giant viruses in the soil samples from the Kutch Desert. Since most giant viruses have been discovered in biofilms in industrial cooling towers, ocean water, and freshwater ponds, we were surprised to find their DNA sequences in soil samples from a seasonally very hot and arid, salty environment.

Evaluating the Quantitative Capabilities of Metagenomic Analysis Software.

DNA sequencing technologies are applied widely and frequently today to describe metagenomes, i.e., microbial communities in environmental or clinical samples, without the need for culturing them. These technologies usually return short (100-300 base-pairs long) DNA reads, and these reads are processed by metagenomic analysis software that assign phylogenetic composition-information to the dataset. Here we evaluate three metagenomic analysis software (AmphoraNet--a webserver implementation of AMPHORA2--, MG-RAST, and MEGAN5) for their capabilities of assigning quantitative phylogenetic information for the data, describing the frequency of appearance of the microorganisms of the same taxa in the sample. The difficulties of the task arise from the fact that longer genomes produce more reads from the same organism than shorter genomes, and some software assign higher frequencies to species with longer genomes than to those with shorter ones. This phenomenon is called the "genome length bias." Dozens of complex artificial metagenome benchmarks can be found in the literature. Because of the complexity of those benchmarks, it is usually difficult to judge the resistance of a metagenomic software to this "genome length bias." Therefore, we have made a simple benchmark for the evaluation of the "taxon-counting" in a metagenomic sample: we have taken the same number of copies of three full bacterial genomes of different lengths, break them up randomly to short reads of average length of 150 bp, and mixed the reads, creating our simple benchmark. Because of its simplicity, the benchmark is not supposed to serve as a mock metagenome, but if a software fails on that simple task, it will surely fail on most real metagenomes. We applied three software for the benchmark. The ideal quantitative solution would assign the same proportion to the three bacterial taxa. We have found that AMPHORA2/AmphoraNet gave the most accurate results and the other two software were under-performers: they counted quite reliably each short read to their respective taxon, producing the typical genome length bias. The benchmark dataset is available at http://pitgroup.org/static/3RandomGenome-100kavg150bps.fna.

Visual analysis of the quantitative composition of metagenomic communities: the AmphoraVizu webserver.

Low-cost DNA sequencing methods have given rise to an enormous development of metagenomics in the past few years. One basic--and difficult--task is the phylogenetic annotation of the metagenomic samples studied. The difficulty comes from the fact that the typical environmental sample contains hundreds of unknown and still uncharacterized microorganisms. There are several possible methods to assign at least partial phylogenetic information to these uncharacterized data. Originally, the 16S ribosomal RNA was used as phylogenetic marker, then genome sequence alignments and similarity measures between the unknown genome and the reference genomes were applied (e.g., in the MEGAN software), and more recently, phylogeny-based methods applying suitable sets of marker genes were suggested (AMPHORA, AMPHORA2, and the webserver implementation AmphoraNet). Here, we present a visual analysis tool that is capable of demonstrating the quantitative relations gained from the output of the AMPHORA2 program or the easy-to-use AmphoraNet webserver. Our web-based tool, the AmphoraVizu webserver, makes the phylogenetic distribution of the metagenomic sample clearly visible by using the native output format of AMPHORA2 or AmphoraNet. The user may set the phylogenetic resolution (i.e., superkingdom, phylum, class, order, family, genus, and species) along with the chart type and will receive the distribution data detailed for all relevant marker genes in the sample. For publication quality results, the chart labels can be customized by the user. The visualization webserver is available at the address http://amphoravizu.pitgroup.org. The AmphoraNet webserver is available at http://amphoranet.pitgroup.org. The open-source version of the AmphoraVizu program is available for download at http://pitgroup.org/apps/amphoravizu/AmphoraVizu.pl.

Unique Patterns in Amino Acid Sequences of Aging-Related Proteins.

Aging has strong genetic components and the list of genes that may regulate the aging process is collected in the GenAge database. There may be characteristic patterns in the amino acid sequences of aging-related proteins that distinguish them from other proteins and this information will lead to a better understanding of the aging process. To test this hypothesis, human protein sequences are extracted from the UniProt database and the relative frequency of every amino acid residue in aging-related proteins and the remaining proteins is calculated. The main observation is that the mean relative frequency of aspartic acid (D) is consistently higher, while the mean relative frequencies of tryptophan (W) and leucine (L) are consistently lower in aging-related proteins compared to the non-aging-related proteins for the human and four examined model organisms. It is also observed that the mean relative frequency of aspartic acid is higher, while the mean relative frequency of tryptophan is lower in pro-longevity proteins compared to anti-longevity proteins in model organisms. Finally, it is found that aging-related proteins tend to be longer than non-aging-related proteins. It is hoped that this analysis initiates further computational and experimental research to explore the underlying mechanisms of these findings.

Alterations of the gut microbiome are associated with epigenetic age acceleration and physical fitness.

Epigenetic clocks can measure aging and predict the incidence of diseases and mortality. Higher levels of physical fitness are associated with a slower aging process and a healthier lifespan. Microbiome alterations occur in various diseases and during the aging process, yet their relation to epigenetic clocks is not explored. To fill this gap, we collected metagenomic (from stool), epigenetic (from blood), and exercise-related data from physically active individuals and, by applying epigenetic clocks, we examined the relationship between gut flora, blood-based epigenetic age acceleration, and physical fitness. We revealed that an increased entropy in the gut microbiome of physically active middle-aged/old individuals is associated with accelerated epigenetic aging, decreased fitness, or impaired health status. We also observed that a slower epigenetic aging and higher fitness level can be linked to altered abundance of some bacterial species often linked to anti-inflammatory effects. Overall our data suggest that alterations in the microbiome can be associated with epigenetic age acceleration and physical fitness.

Blood Test-Based Age Acceleration Is Inversely Associated with High-Volume Sports Activity.

PURPOSE: We develop blood test-based aging clocks and examine how these clocks reflect high-volume sports activity. METHODS: We use blood tests and body metrics data of 421 Hungarian athletes and 283 age-matched controls (mean age, 24.1 and 23.9 yr, respectively), the latter selected from a group of healthy Caucasians of the National Health and Nutrition Examination Survey (NHANES) to represent the general population ( n = 11,412). We train two age prediction models (i.e., aging clocks) using the NHANES dataset: the first model relies on blood test parameters only, whereas the second one additionally incorporates body measurements and sex. RESULTS: We find lower age acceleration among athletes compared with the age-matched controls with a median value of -1.7 and 1.4 yr, P < 0.0001. BMI is positively associated with age acceleration among the age-matched controls ( r = 0.17, P < 0.01) and the unrestricted NHANES population ( r = 0.11, P < 0.001). We find no association between BMI and age acceleration within the athlete dataset. Instead, age acceleration is positively associated with body fat percentage ( r = 0.21, P < 0.05) and negatively associated with skeletal muscle mass (Pearson r = -0.18, P < 0.05) among athletes. The most important blood test features in age predictions were serum ferritin, mean cell volume, blood urea nitrogen, and albumin levels. CONCLUSIONS: We develop and apply blood test-based aging clocks to adult athletes and healthy controls. The data suggest that high-volume sports activity is associated with slowed biological aging. Here, we propose an alternative, promising application of routine blood tests.

Association of Baseline Tumor-Specific Neoantigens and CD8+ T-Cell Infiltration With Immune-Related Adverse Events Secondary to Immune Checkpoint Inhibitors.

PURPOSE: Recent evidence has shown that higher tumor mutational burden strongly correlates with an increased risk of immune-related adverse events (irAEs). By using an integrated multiomics approach, we further studied the association between relevant tumor immune microenvironment (TIME) features and irAEs. METHODS: Leveraging the US Food and Drug Administration Adverse Event Reporting System, we extracted cases of suspected irAEs to calculate the reporting odds ratios (RORs) of irAEs for cancers treated with immune checkpoint inhibitors (ICIs). TIME features for 32 cancer types were calculated on the basis of the cancer genomic atlas cohorts and indirectly correlated with each cancer's ROR for irAEs. A separate ICI-treated cohort of non-small-cell lung cancer (NSCLC) was used to evaluate the correlation between tissue-based immune markers (CD8+, PD-1/L1+, FOXP3+, tumor-infiltrating lymphocytes [TILs]) and irAE occurrence. RESULTS: The analysis of 32 cancers and 33 TIME features demonstrated a significant association between irAE RORs and the median number of base insertions and deletions (INDEL), neoantigens (r = 0.72), single-nucleotide variant neoantigens (r = 0.67), and CD8+ T-cell fraction (r = 0.51). A bivariate model using the median number of INDEL neoantigens and CD8 T-cell fraction had the highest accuracy in predicting RORs (adjusted r2 = 0.52, P = .002). Immunoprofile assessment of 156 patients with NSCLC revealed a strong trend for higher baseline median CD8+ T cells within patients' tumors who experienced any grade irAEs. Using machine learning, an expanded ICI-treated NSCLC cohort (n = 378) further showed a treatment duration-independent association of an increased proportion of high TIL (>median) in patients with irAEs (59.7% v 44%, P = .005). This was confirmed by using the Fine-Gray competing risk approach, demonstrating higher baseline TIL density (>median) associated with a higher cumulative incidence of irAEs (P = .028). CONCLUSION: Our findings highlight a potential role for TIME features, specifically INDEL neoantigens and baseline-immune infiltration, in enabling optimal irAE risk stratification of patients.

Age-associated clonal B cells drive B cell lymphoma in mice.

Although cancer is an age-related disease, how the processes of aging contribute to cancer progression is not well understood. In this study, we uncovered how mouse B cell lymphoma develops as a consequence of a naturally aged system. We show here that this malignancy is associated with an age-associated clonal B cell (ACBC) population that likely originates from age-associated B cells. Driven by c-Myc activation, promoter hypermethylation and somatic mutations, IgM+ ACBCs clonally expand independently of germinal centers and show increased biological age. ACBCs become self-sufficient and support malignancy when transferred into young recipients. Inhibition of mTOR or c-Myc in old mice attenuates pre-malignant changes in B cells during aging. Although the etiology of mouse and human B cell lymphomas is considered distinct, epigenetic changes in transformed mouse B cells are enriched for changes observed in human B cell lymphomas. Together, our findings characterize the spontaneous progression of cancer during aging through both cell-intrinsic and microenvironmental changes and suggest interventions for its prevention.

TIME-seq reduces time and cost of DNA methylation measurement for epigenetic clock construction.

Epigenetic 'clocks' based on DNA methylation have emerged as the most robust and widely used aging biomarkers, but conventional methods for applying them are expensive and laborious. Here we develop tagmentation-based indexing for methylation sequencing (TIME-seq), a highly multiplexed and scalable method for low-cost epigenetic clocks. Using TIME-seq, we applied multi-tissue and tissue-specific epigenetic clocks in over 1,800 mouse DNA samples from eight tissue and cell types. We show that TIME-seq clocks are accurate and robust, enriched for polycomb repressive complex 2-regulated loci, and benchmark favorably against conventional methods despite being up to 100-fold less expensive. Using dietary treatments and gene therapy, we find that TIME-seq clocks reflect diverse interventions in multiple tissues. Finally, we develop an economical human blood clock (R > 0.96, median error = 3.39 years) in 1,056 demographically representative individuals. These methods will enable more efficient epigenetic clock measurement in larger-scale human and animal studies.

Intersection clock reveals a rejuvenation event during human embryogenesis.

Recent research revealed a rejuvenation event during early development of mice. Here, by examining epigenetic age dynamics of human embryogenesis, we tested whether a similar event exists in humans. For this purpose, we developed an epigenetic clock method, the intersection clock, that utilizes bisulfite sequencing in a way that maximizes the use of informative CpG sites with no missing clock CpG sites in test samples and applied it to human embryo development data. We observed no changes in the predicted epigenetic age between cleavage stage and blastocyst stage embryos; however, a significant decrease was observed between blastocysts and cells representing the epiblast. Additionally, by applying the intersection clock to datasets spanning pre and postimplantation, we found no significant change in the epigenetic age during preimplantation stages; however, the epigenetic age of postimplantation samples was lower compared to the preimplantation stages. We further investigated the epigenetic age of primed (representing early postimplantation) and naïve (representing preimplantation) pluripotent stem cells and observed that in all cases the epigenetic age of primed cells was significantly lower than that of naïve cells. Together, our data suggest that human embryos are rejuvenated during early embryogenesis. Hence, the rejuvenation event is conserved between the mouse and human, and it occurs around the gastrulation stage in both species. Beyond this advance, the intersection clock opens the way for other epigenetic age studies based on human bisulfite sequencing datasets as opposed to methylation arrays.

Multi-omic rejuvenation and life span extension on exposure to youthful circulation.

Heterochronic parabiosis (HPB) is known for its functional rejuvenation effects across several mouse tissues. However, its impact on biological age and long-term health is unknown. Here we performed extended (3-month) HPB, followed by a 2-month detachment period of anastomosed pairs. Old detached mice exhibited improved physiological parameters and lived longer than control isochronic mice. HPB drastically reduced the epigenetic age of blood and liver based on several clock models using two independent platforms. Remarkably, this rejuvenation effect persisted even after 2 months of detachment. Transcriptomic and epigenomic profiles of anastomosed mice showed an intermediate phenotype between old and young, suggesting a global multi-omic rejuvenation effect. In addition, old HPB mice showed gene expression changes opposite to aging but akin to several life span-extending interventions. Altogether, we reveal that long-term HPB results in lasting epigenetic and transcriptome remodeling, culminating in the extension of life span and health span.

Downregulation of transposable elements extends lifespan in Caenorhabditis elegans.

Mobility of transposable elements (TEs) frequently leads to insertional mutations in functional DNA regions. In the potentially immortal germline, TEs are effectively suppressed by the Piwi-piRNA pathway. However, in the genomes of ageing somatic cells lacking the effects of the pathway, TEs become increasingly mobile during the adult lifespan, and their activity is associated with genomic instability. Whether the progressively increasing mobilization of TEs is a cause or a consequence of ageing remains a fundamental problem in biology. Here we show that in the nematode Caenorhabditis elegans, the downregulation of active TE families extends lifespan. Ectopic activation of Piwi proteins in the soma also promotes longevity. Furthermore, DNA N6-adenine methylation at TE stretches gradually rises with age, and this epigenetic modification elevates their transcription as the animal ages. These results indicate that TEs represent a novel genetic determinant of ageing, and that N6-adenine methylation plays a pivotal role in ageing control.

Biological age is increased by stress and restored upon recovery.

Aging is classically conceptualized as an ever-increasing trajectory of damage accumulation and loss of function, leading to increases in morbidity and mortality. However, recent in vitro studies have raised the possibility of age reversal. Here, we report that biological age is fluid and exhibits rapid changes in both directions. At epigenetic, transcriptomic, and metabolomic levels, we find that the biological age of young mice is increased by heterochronic parabiosis and restored following surgical detachment. We also identify transient changes in biological age during major surgery, pregnancy, and severe COVID-19 in humans and/or mice. Together, these data show that biological age undergoes a rapid increase in response to diverse forms of stress, which is reversed following recovery from stress. Our study uncovers a new layer of aging dynamics that should be considered in future studies. The elevation of biological age by stress may be a quantifiable and actionable target for future interventions.

DNA methylation clock DNAmFitAge shows regular exercise is associated with slower aging and systemic adaptation.

DNAmPhenoAge, DNAmGrimAge, and the newly developed DNAmFitAge are DNA methylation (DNAm)-based biomarkers that reflect the individual aging process. Here, we examine the relationship between physical fitness and DNAm-based biomarkers in adults aged 33-88 with a wide range of physical fitness (including athletes with long-term training history). Higher levels of VO2max (ρ = 0.2, p = 6.4E - 4, r = 0.19, p = 1.2E - 3), Jumpmax (p = 0.11, p = 5.5E - 2, r = 0.13, p = 2.8E - 2), Gripmax (ρ = 0.17, p = 3.5E - 3, r = 0.16, p = 5.6E - 3), and HDL levels (ρ = 0.18, p = 1.95E - 3, r = 0.19, p = 1.1E - 3) are associated with better verbal short-term memory. In addition, verbal short-term memory is associated with decelerated aging assessed with the new DNAm biomarker FitAgeAcceleration (ρ: - 0.18, p = 0.0017). DNAmFitAge can distinguish high-fitness individuals from low/medium-fitness individuals better than existing DNAm biomarkers and estimates a younger biological age in the high-fit males and females (1.5 and 2.0 years younger, respectively). Our research shows that regular physical exercise contributes to observable physiological and methylation differences which are beneficial to the aging process. DNAmFitAge has now emerged as a new biological marker of quality of life.

Emerging rejuvenation strategies-Reducing the biological age.

Several interventions have recently emerged that were proposed to reverse rather than just attenuate aging, but the criteria for what it takes to achieve rejuvenation remain controversial. Distinguishing potential rejuvenation therapies from other longevity interventions, such as those that slow down aging, is challenging, and these anti-aging strategies are often referred to interchangeably. We suggest that the prerequisite for a rejuvenation intervention is a robust, sustained, and systemic reduction in biological age, which can be assessed by biomarkers of aging, such as epigenetic clocks. We discuss known and putative rejuvenation interventions and comparatively analyze them to explore underlying mechanisms.