N-glycan profiling of tissue samples to aid breast cancer subtyping.

Breast cancer is a highly heterogeneous disease. Its intrinsic subtype classification for diagnosis and choice of therapy traditionally relies on the presence of characteristic receptors. Unfortunately, this classification is often not sufficient for precise prediction of disease prognosis and treatment efficacy. The N-glycan profiles of 145 tumors and 10 healthy breast tissues were determined using Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry. The tumor samples were classified into Mucinous, Lobular, No-Special-Type, Human Epidermal Growth Factor 2 + , and Triple-Negative Breast Cancer subtypes. Statistical analysis was conducted using the reproducibility-optimized test statistic software package in R, and the Wilcoxon rank sum test with continuity correction. In total, 92 N-glycans were detected and quantified, with 59 consistently observed in over half of the samples. Significant variations in N-glycan signals were found among subtypes. Mucinous tumor samples exhibited the most distinct changes, with 28 significantly altered N-glycan signals. Increased levels of tri- and tetra-antennary N-glycans were notably present in this subtype. Triple-Negative Breast Cancer showed more N-glycans with additional mannose units, a factor associated with cancer progression. Individual N-glycans differentiated Human Epidermal Growth Factor 2 + , No-Special-Type, and Lobular cancers, whereas lower fucosylation and branching levels were found in N-glycans significantly increased in Luminal subtypes (Lobular and No-Special-Type tumors). Clinically normal breast tissues featured a higher abundance of signals corresponding to N-glycans with bisecting moiety. This research confirms that histologically distinct breast cancer subtypes have a quantitatively unique set of N-glycans linked to clinical parameters like tumor size, proliferative rate, lymphovascular invasion, and metastases to lymph nodes. The presented results provide novel information that N-glycan profiling could accurately classify human breast cancer samples, offer stratification of patients, and ongoing disease monitoring.

Intrinsically disordered proteins and liquid-liquid phase separation in SARS-CoV-2 interactomes.

This paper discusses the properties of proteins and their relations in the interactomes of the selected subsets of SARS-CoV-2 proteome-the membrane protein, nonstructural proteins, and, finally, full proteome. Protein disorder according to several measures, liquid-liquid phase separation probabilities, and protein node degrees in the interaction networks were singled out as the features of interest. Additionally, viral interactomes were combined with the interactome of human lung tissue so as to examine if the new connections in the resulting viral-host interactome are linked to protein disorder. Correlation analysis shows that there is no clear relationship between raw features of interest, whereas there is a positive correlation between the protein disorder and its neighborhood mean disorder. There are also indications that highly connected viral hubs tend to be on average more ordered than proteins with a small number of connections. This is in contrast to previous similar studies conducted on eukaryotic interactomes and possibly raises new questions in research on viral interactomes.

Assessing the effect of compounds from plantar foot sweat, nesting material, and urine on social behavior in male mice, Mus musculus.

Home cage aggression causes poor welfare in male laboratory mice and reduces data quality. One of the few proven strategies to reduce aggression involves preserving used nesting material at cage change. Volatile organic compounds from the nesting material and several body fluids not only correlate with less home cage aggression, but with more affiliative allo-grooming behavior. To date, these compounds have not been tested for a direct influence on male mouse social behavior. This study aimed to determine if 4 previously identified volatile compounds impact home cage interactions. A factorial design was used with cages equally split between C57BL/6N and SJL male mice (N = 40). Treatments were randomly assigned across cages and administered by spraying one compound solution on each cage's nesting material. Treatments were refreshed after day 3 and during cage change on day 7. Home cage social behavior was observed throughout the study week and immediately after cage change. Several hours after cage change, feces were collected from individual mice to measure corticosterone metabolites as an index of social stress. Wound severity was also assessed after euthanasia. Measures were analyzed with mixed models. Compound treatments did not impact most study measures. For behavior, SJL mice performed more aggression and submission, and C57BL/6N mice performed more allo-grooming. Wound severity was highest in the posterior region of both strains, and the middle back region of C57BL/6N mice. Posterior wounding also increased with more observed aggression. Corticosterone metabolites were higher in C57BL/6N mice and in mice treated with 3,4-dimethyl-1,2-cyclopentanedione with more wounding. These data confirm previous strain patterns in social behavior and further validates wound assessment as a measure of escalated aggression. The lack of observed treatment effects could be due to limitations in the compound administration procedure and/or the previous correlation study, which is further discussed.

Fractionation and characterization of sialyl linkage isomers of serum N-glycans by CE-MS.

Structural isomers of sialylated N-glycans contribute to the diversity of the N-glycome and to a range of biological functions. Sialyl linkage isomers can be readily distinguished by mass spectrometry with mass differences between α2,3- and α2,6-linkages generated by a two-step sialic acid linkage-specific alkylamidation. To improve the identification of N-glycans from complex mixtures, we added a delactonization step after the first alkylamidation step, which regenerates negatively charged carboxylic acids on α2,3-sialic acids. N-glycan isomers with α2,3-sialic acids are then fractionated by ion-exchange chromatography prior to the second alkylamidation step. With this modified alkylamidation method, sialylated N-glycans were enriched and stabilized for structural characterization by capillary electrophoresis-mass spectrometry and tandem mass spectrometry. We identified 52 sialylated N-glycan structures, including 107 linkage isomers, in human serum and confirmed the presence of positional isomers of specific sialyl linkage isomers. Due to the reduced sample complexity after ion-exchange fractionation and CE separation, substructural features of N-glycans were rapidly evaluated and included core- and antenna-fucosylation and poly-lactosamine.

A graphical representation of glycan heterogeneity.

A substantial shortcoming of large-scale datasets is often the inability to easily represent and visualize key features. This problem becomes acute when considering the increasing technical ability to profile large numbers of glycopeptides and glycans in recent studies. Here, we describe a simple, concise graphical representation intended to capture the microheterogeneity associated with glycan modification at specific sites. We illustrate this method by showing visual representations of the glycans and glycopeptides from a variety of species. The graphical representation presented allows one to easily discern the compositions of all glycans, similarities and differences of modifications found in different samples and, in the case of N-linked glycans, the initial steps in the biosynthetic pathway.

Virtual screening, drug-likeness analysis, and molecular docking study of potential severe acute respiratory syndrome coronavirus 2 main protease inhibitors.

Due to the length of time required to develop specific antiviral agents, the World Health Organization adopted the strategy of repurposing existing medications to treat Coronavirus disease 2019 infection. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease is possible biological target for potential antiviral drugs. We selected various compounds from PubChem database based on the structure of main protease inhibitors in Protein Data Bank database. Ten compounds showed nontumorigenic and nonmutagenic potential and met Egan's and Lipinski's rules. Molecular docking analysis was performed using AutoDock Vina software. Based on number and type of key binding interactions, as well as docking scores, we selected compounds 6, 8, and 17 that demonstrated the highest binding affinity for the target protein. Molecular dynamics simulations were then carried out on the protein-top docked ligand complexes which were subjected to molecular mechanics/generalized Born and surface area calculations. The molecular dynamics simulation results indicated that protein-top docked ligand complexes showed good conformational stability. Among analyzed molecules, compound 17 emerged as the best in silico hit based on the docking score, MM/GBSA binding energy and MD results.

Burnout Among Private Security Staff in Serbia: A Multicentic Cross-Sectional Study.

Background: Burnout is a special a state of physical or emotional exhaustion that also involves a sense of reduced accomplishment and loss of personal identity. Objectives: To evaluate the predictors of burnout among work staff in the seven private agencies for support and defense of persons and their property. Material and Method: We performed a multicentric cross-sectional study that involved representative sample of working staff from Agencies of Private Security in Central Serbia. Burnout was assessed using Maslach Burnout Inventory- (MBI)-Human Services Survey. Results: A total number of participants were 353 (330 men and 23 women). Measured level of burnout as assessed by high emotional exhaustion, high depersonalization, and low personal accomplishment was 66.3, 82.4, and 13.4%, respectively. We identified that female gender, younger age, shorter work experience, working in shifts, working 12 h a day and more than 8-12 h a day as well as dissatisfaction with working conditions. Work in shifts, working 12 h a day and more than 8-12 h a day and dissatisfied with dissatisfaction with working conditions significantly increase the risk of total burnout. Conclusion: Our results showed that significant predictors for the development of burnout syndrome were female gender, younger age, shorter work experience, working in shifts, as well as dissatisfaction with working conditions.

Compounds from plantar foot sweat, nesting material, and urine show strain patterns associated with agonistic and affiliative behaviors in group housed male mice, Mus musculus.

Excessive home cage aggression often results in severe injury and subsequent premature euthanasia of male laboratory mice. Aggression can be reduced by transferring used nesting material during cage cleaning, which is thought to contain aggression appeasing odors from the plantar sweat glands. However, neither the composition of plantar sweat nor the deposits on used nesting material have been evaluated. The aims of this study were to (1) identify and quantify volatile compounds deposited in the nest site and (2) determine if nest and sweat compounds correlate with social behavior. Home cage aggression and affiliative behavior were evaluated in 3 strains: SJL, C57BL/6N, and A/J. Individual social rank was assessed via the tube test, because ranking may influence compound levels. Sweat and urine from the dominant and subordinate mouse in each cage, plus cage level nest samples were analyzed for volatile compound content using gas chromatography-mass spectrometry. Behavior data and odors from the nest, sweat, and urine were statistically analyzed with separate principal component analyses (PCA). Significant components, from each sample analysis, and strain were run in mixed models to test if odors were associated with behavior. Aggressive and affiliative behaviors were primarily impacted by strain. However, compound PCs were also impacted by strain, showing that strain accounts for any relationship between odors and behavior. C57BL/6N cages displayed the most allo-grooming behavior and had high scores on sweat PC1. SJL cages displayed the most aggression, with high scores on urine PC2 and low scores on nest PC1. These data show that certain compounds in nesting material, urine, and sweat display strain specific patterns which match strain specific behavior patterns. These results provide preliminary information about the connection between home cage compounds and behavior. Salient compounds will be candidates for future controlled studies to determine their direct effect on mouse social behavior.

N-Glycome changes reflecting resistance to platinum-based chemotherapy in ovarian cancer.

A number of studies have reported aberrant glycosylation in connection with malignancy. Our investigation further expands on this topic through the examination of N-glycans, which could be associated with the resistance of advanced stage, high-grade non-mucinous ovarian cancer to platinum/taxane based chemotherapy. We used tissue samples of 83 ovarian cancer patients, randomly divided into two independent cohorts (basic and validation). Both groups involved either cases with/without postoperative tumor residue or the cases determined either resistant or sensitive to this chemotherapy. In the validation cohort, preoperative serum samples were also available. N-glycans released from tumors and sera were permethylated and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The MS analysis yielded a consecutive detection of 68 (tissue) and 63 (serum) N-glycan spectral signals. Eight of these were found to be differentially abundant in tissues of both independent cohorts including the cases with a postoperative cancer residue. One of these glycans was detected as differentially abundant in sera of the validation cohort. No statistically significant differences in intensities due to the same N-glycans were found in the cases without postoperative macroscopic residues in either the basic or validation cohort. From the biochemical point of view, the statistically significant N-glycans correspond to the structures carrying bisecting (terminal) GlcNAc residue and tetra-antennary structures with sialic acid and/or fucose residues. Among them, six tissue N-glycans could be considered potential markers connected with a resistance to chemotherapy in ovarian cancer patients. The prediction of primary resistance to standard chemotherapy may identify the group of patients suitable for alternative treatment strategies. SIGNIFICANCE: Drug resistance has become a major impediment to a successful treatment of patients with advanced ovarian cancer. The glycomic measurements related to cancer are becoming increasingly popular in identification of the key molecules as potential diagnostic and prognostic indicators. Our report deals with identification of differences in N-glycosylation of proteins in tissue and serum samples from the individuals showing sensitivity or resistance to platinum/taxane-based chemotherapy. The detection sensitivity to chemotherapy is vitally important for these patients.

Exosome-Mediated Crosstalk between Keratinocytes and Macrophages in Cutaneous Wound Healing.

Bidirectional cell-cell communication involving exosome-borne cargo such as miRNA has emerged as a critical mechanism for wound healing. Unlike other shedding vesicles, exosomes selectively package miRNA by SUMOylation of heterogeneous nuclear ribonucleoproteinA2B1 (hnRNPA2B1). In this work, we elucidate the significance of exosome in keratinocyte-macrophage crosstalk following injury. Keratinocyte-derived exosomes were genetically labeled with GFP-reporter (Exoκ-GFP) using tissue nanotransfection (TNT), and they were isolated from dorsal murine skin and wound-edge tissue by affinity selection using magnetic beads. Surface N-glycans of Exoκ-GFP were also characterized. Unlike skin exosome, wound-edge Exoκ-GFP demonstrated characteristic N-glycan ions with abundance of low-base-pair RNA and was selectively engulfed by wound macrophages (ωmϕ) in granulation tissue. In vitro addition of wound-edge Exoκ-GFP to proinflammatory ωmϕ resulted in conversion to a proresolution phenotype. To selectively inhibit miRNA packaging within Exoκ-GFPin vivo, pH-responsive keratinocyte-targeted siRNA-hnRNPA2B1 functionalized lipid nanoparticles (TLNPκ) were designed with 94.3% encapsulation efficiency. Application of TLNPκ/si-hnRNPA2B1 to the murine dorsal wound-edge significantly inhibited expression of hnRNPA2B1 by 80% in epidermis compared to the TLNPκ/si-control group. Although no significant difference in wound closure or re-epithelialization was observed, the TLNPκ/si-hnRNPA2B1 treated group showed a significant increase in ωmϕ displaying proinflammatory markers in the granulation tissue at day 10 post-wounding compared to the TLNPκ/si-control group. Furthermore, TLNPκ/si-hnRNPA2B1 treated mice showed impaired barrier function with diminished expression of epithelial junctional proteins, lending credence to the notion that unresolved inflammation results in leaky skin. This work provides insight wherein Exoκ-GFP is recognized as a major contributor that regulates macrophage trafficking and epithelial barrier properties postinjury.

Glycoproteomic Analysis of Human Urinary Exosomes.

Exosomes represent a class of secreted biological vesicles, which have recently gained attention due to their function as intertissue and interorganism transporters of genetic materials, small molecules, lipids, and proteins. Although the protein constituents of these exosomes are often glycosylated, a large-scale characterization of the glycoproteome has not yet been completed. This study identified 3144 unique glycosylation events belonging to 378 glycoproteins and 604 unique protein sites of glycosylation. With these data, we investigated the level of glycan microheterogeneity within the urinary exosomes, finding on average 5.9 glycans per site. The glycan family abundance on individual proteins showed subtle differences, providing an additional level of molecular characterization compared to the unmodified proteome. Finally, we show protein site-specific changes in regard to the common urinary glycoprotein, uromodulin. While uromodulin is an individual case, these same site-specific analyses provide a way forward for developing diagnostic glycoprotein biomarkers with urine as a noninvasive biological fluid. This study represents an important first step in understanding the functional urinary glycoproteome.

Volatile fatty acid and aldehyde abundances evolve with behavior and habitat temperature in Sceloporus lizards.

Animal signals evolve by striking a balance between the need to convey information through particular habitats and the limitations of what types of signals can most easily be produced and perceived. Here, we present new results from field measures of undisturbed behavior and biochemical analyses of scent marks from 12 species of Sceloporus lizards to explore whether evolutionary changes in chemical composition are better predicted by measures of species behavior, particularly those associated with visual displays, chemoreception, and locomotion, or by measures of habitat climate (precipitation and temperature). We found that more active lizard species used fewer compounds in their volatile scent marks, perhaps conveying less specific information about individual and species identity. Scent marks from more active lizard species also had higher proportions of saturated fatty acids, and the evolution of these compounds has been tracking the phylogeny closely as we would expect for a metabolic byproduct. In contrast, the proportions of unsaturated fatty acids were better explained by evolutionary shifts in habitat temperature (and not precipitation), with species in warmer climates using almost no volatile unsaturated fatty acids. The proportion of aldehydes was explained by both behavior and environment, decreasing with behavioral activity and increasing with habitat temperature. Our results highlight the evolutionary flexibility of complex chemical signals, with different chemical compounds responding to different elements of the selective landscape over evolutionary time.

Structural Identification, Synthesis and Biological Activity of Two Volatile Cyclic Dipeptides in a Terrestrial Vertebrate.

Single substances within complex vertebrate chemical signals could be physiologically or behaviourally active. However, the vast diversity in chemical structure, physical properties and molecular size of semiochemicals makes identifying pheromonally active compounds no easy task. Here, we identified two volatile cyclic dipeptides, cyclo(L-Leu-L-Pro) and cyclo(L-Pro-L-Pro), from the complex mixture of a chemical signal in terrestrial vertebrates (lizard genus Sceloporus), synthesised one of them and investigated their biological activity in male intra-specific communication. In a series of behavioural trials, lizards performed more chemosensory behaviour (tongue flicks, lip smacks and substrate lickings) when presented with the synthesised cyclo(L-Pro-L-Pro) chemical blend, compared to the controls, the cyclo(L-Leu-L-Pro) blend, or a combined blend with both cyclic dipeptides. The results suggest a potential semiochemical role of cyclo(L-Pro-L-Pro) and a modulating effect of cyclo(L-Leu-L-Pro) that may depend on the relative concentration of both compounds in the chemical signal. In addition, our results stress how minor compounds in complex mixtures can produce a meaningful behavioural response, how small differences in structural design are crucial for biological activity, and highlight the need for more studies to determine the complete functional landscape of biologically relevant compounds.

Charge Detection Mass Spectrometry Measurements of Exosomes and other Extracellular Particles Enriched from Bovine Milk.

The masses of particles in a bovine milk extracellular vesicle (EV) preparation enriched for exosomes were directly determined for the first time by charge detection mass spectrometry (CDMS). In CDMS, both the mass-to-charge ratio (m/z) and z are determined simultaneously for individual particles, enabling mass determinations for particles that are far beyond the mass limit (∼1.0 MDa) of conventional mass spectrometry (MS). Particle masses and charges span a wide range from m ∼ 2 to ∼90 MDa and z ∼ 50 to ∼1300 e (elementary charges) and are highly dependent upon the conditions used to extract and isolate the EVs. EV particles span a continuum of masses, reflecting the highly heterogeneous nature of these samples. However, evidence for unique populations of particles is obtained from correlation of the charges and masses. An analysis that uses a two-dimensional Gaussian model, provides evidence for six families of particles, four of which having masses in the range expected for exosomes. Complementary proteomics measurements and electron microscopy (EM) imaging are used to further characterize the EVs and confirm that these samples have been enriched in exosomes. The ability to characterize such extremely heterogeneous mixtures of large particles with rapid, sensitive, and high-resolution MS techniques is critical to ongoing analytical efforts to separate and purify exosomes and exosome subpopulations. Direct measurement of each particle's mass and charge is a new means of characterizing the physical and chemical properties of exosomes and other EVs.

Beta-caryophyllene enhances wound healing through multiple routes.

Beta-caryophyllene is an odoriferous bicyclic sesquiterpene found in various herbs and spices. Recently, it was found that beta-caryophyllene is a ligand of the cannabinoid receptor 2 (CB2). Activation of CB2 will decrease pain, a major signal for inflammatory responses. We hypothesized that beta-caryophyllene can affect wound healing by decreasing inflammation. Here we show that cutaneous wounds of mice treated with beta-caryophyllene had enhanced re-epithelialization. The treated tissue showed increased cell proliferation and cells treated with beta-caryophyllene showed enhanced cell migration, suggesting that the higher re-epithelialization is due to enhanced cell proliferation and cell migration. The treated tissues also had up-regulated gene expression for hair follicle bulge stem cells. Olfactory receptors were not involved in the enhanced wound healing. Transient Receptor Potential channel genes were up-regulated in the injured skin exposed to beta-caryophyllene. Interestingly, there were sex differences in the impact of beta- caryophyllene as only the injured skin of female mice had enhanced re-epithelialization after exposure to beta-caryophyllene. Our study suggests that chemical compounds included in essential oils have the capability to improve wound healing, an effect generated by synergetic impacts of multiple pathways.

Experimental evidence that symbiotic bacteria produce chemical cues in a songbird.

Symbiotic microbes that inhabit animal scent glands can produce volatile compounds used as chemical signals by the host animal. Though several studies have demonstrated correlations between scent gland bacterial community structure and host animal odour profiles, none have systematically demonstrated a causal relationship. In birds, volatile compounds in preen oil secreted by the uropygial gland serve as chemical cues and signals. Here, we tested whether manipulating the uropygial gland microbial community affects chemical profiles in the dark-eyed junco (Junco hyemalis). We found an effect of antibiotic treatment targeting the uropygial gland on both bacterial and volatile profiles. In a second experiment, we cultured bacteria from junco preen oil, and found that all of the cultivars produced at least one volatile compound common in junco preen oil, and that most cultivars produced multiple preen oil volatiles. In both experiments, we identified experimentally generated patterns in specific volatile compounds previously shown to predict junco reproductive success. Together, our data provide experimental support for the hypothesis that symbiotic bacteria produce behaviourally relevant volatile compounds within avian chemical cues and signals.

In-Depth Compositional and Structural Characterization of N-Glycans Derived from Human Urinary Exosomes.

The study of exosomes has become increasingly popular due to their potentially important biological roles. Urine can be used as an effective source of exosomes for noninvasive investigations into the pathophysiological states of the urinary system, but first, detailed characterization of exosomal components in healthy individuals is essential. Here, we significantly extend the number of N-glycan compositions, including sulfated species, identified from urinary exosomes and determine the sialic acid linkages for many of those compositions. Capillary electrophoresis-mass spectrometry (CE-MS), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), and capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to identify N-glycan and sulfated N-glycan compositions. Second, because the alteration of sialylation patterns has been previously implicated in various disease states, ion-exchange chromatography, microfluidic capillary electrophoresis (CE), and MALDI-MS were adopted to resolve positional isomers of sialic acids. Structures of the sialyl-linkage isomers were assigned indirectly through α2-3 sialidase treatment and sialic acid linkage-specific alkylamidation (SALSA). In total, we have identified 219 N-glycan structures that include 175 compositions, 64 sialic acid linkage isomers, 26 structural isomers, and 27 sulfated glycans.

Highly Sensitive O-Glycan Profiling for Human Serum Proteins Reveals Gender-Dependent Changes in Colorectal Cancer Patients.

A newly developed microscale protocol for profiling serum O-glycans has been validated here with multiple serum samples obtained from different cohorts of colorectal cancer patients. The simultaneous cleavage and permethylation steps in this procedure preserve the integrity of released minor O-glycans, so that 39 O-linked oligosaccharides could be reliably recorded in a profile. This is far more detected components than shown in any previous studies. The analytical results were further subjected to a battery of statistical tests. Our O-glycan compositions compare favorably with the previous results obtained with solid tumors and cancer cell lines, suggesting that smaller circulatory mucins protruding into the blood circulation may be one source of O-glycans that we observe in the serum samples. While the control vs cancer statistical comparisons generally agree with the expected glycosylation trends, the comparisons of male vs female subjects have led to some surprising results for which we do not have a ready explanation due to lack of any literature describing hormonal control of O-glycosylation. Our results thus underscore the necessity of applying new analytical technologies to clinically interesting sample sets.

Analytical Scheme Leading to Integrated High-Sensitivity Profiling of Glycosphingolipids Together with N- and O-Glycans from One Sample.

Glycoconjugates are directly or indirectly involved in many biological processes. Due to their complex structures, the structural elucidation of glycans and the exploration of their role in biological systems have been challenging. Glycan pools generated through release from glycoprotein or glycolipid mixtures can often be very complex. For the sake of procedural simplicity, many glycan profiling studies choose to concentrate on a single class of glycoconjugates. In this paper, we demonstrate it feasible to cover glycosphingolipids, N-glycans, and O-glycans isolated from the same sample. Small volumes of human blood serum and ascites fluid as well as small mouse brain tissue samples are sufficient to profile sequentially glycans from all three classes of glycoconjugates and even positively identify some mixture components through MALDI-MS and LC-ESI-MS. The results show that comprehensive glycan profiles can be obtained from the equivalent of 500-µg protein starting material or possibly less. These methodological improvements can help accelerating future glycomic comprehensive studies, especially for precious clinical samples. Graphical Abstract Outline of glycan profiling procedures.

Structural disorder of plasmid-encoded proteins in Bacteria and Archaea.

BACKGROUND: In the last decade and a half it has been firmly established that a large number of proteins do not adopt a well-defined (ordered) structure under physiological conditions. Such intrinsically disordered proteins (IDPs) and intrinsically disordered (protein) regions (IDRs) are involved in essential cell processes through two basic mechanisms: the entropic chain mechanism which is responsible for rapid fluctuations among many alternative conformations, and molecular recognition via short recognition elements that bind to other molecules. IDPs possess a high adaptive potential and there is special interest in investigating their involvement in organism evolution. RESULTS: We analyzed 2554 Bacterial and 139 Archaeal proteomes, with a total of 8,455,194 proteins for disorder content and its implications for adaptation of organisms, using three disorder predictors and three measures. Along with other findings, we revealed that for all three predictors and all three measures (1) Bacteria exhibit significantly more disorder than Archaea; (2) plasmid-encoded proteins contain considerably more IDRs than proteins encoded on chromosomes (or whole genomes) in both prokaryote superkingdoms; (3) plasmid proteins are significantly more disordered than chromosomal proteins only in the group of proteins with no COG category assigned; (4) antitoxin proteins in comparison to other proteins, are the most disordered (almost double) in both Bacterial and Archaeal proteomes; (5) plasmidal proteins are more disordered than chromosomal proteins in Bacterial antitoxins and toxin-unclassified proteins, but have almost the same disorder content in toxin proteins. CONCLUSION: Our results suggest that while disorder content depends on genome and proteome characteristics, it is more influenced by functional engagements than by gene location (on chromosome or plasmid).