Sequence-based prediction of the effects of histones H1 post-translational modifications: impact on the features related to the function.

Post-translational modifications modulate histones H1 activity but their impact on proteins features was not studied so far. Therefore, this work was intended to answer how the most common modifications, i.e. acetylation, methylation, phosphorylation and ubiquitination, can influence on histones H1 to alter their physicochemical and molecular properties. Investigations were done with the use of sequence-based predictors trained on various protein features. Because a full set of histones H1 modifications is not included in the databases of histone proteins, the survey was performed on the human, animals, plants, fungi and protist sequences selected from UniProtKB/Swiss-Prot database. Quantitative proportions of modifications were similar between the groups of organisms (CV = 0.11) but different within the group (p < 0.05). The effects of modifications were evaluated with the use of mutated sequences obtained through the substitution of modified residue of Lys, Ser and Thr by a neutral residue of the Ala. An advantage of deleterious mutations at the sites of acetylation, methylation and ubiquitination over the sites of phosphorylation (p < 0.05) indicate that this modification have more redundant character. Modifications evoke an increase of protein solubility and stability as well as acceleration of folding kinetics and a weaken of binding affinity. Besides, they also maintain a higher extent of intrinsic structural disorder. The obtained results prove that modifications should be perceived as relevant factors influencing physicochemical features determining molecular properties. Thus, histones H1 functioning is strictly correlated with the status of modifications.Communicated by Ramaswamy H. Sarma.

PD-1 and PD-L1 Expression Levels as a Potential Biomarker of Chronic Rhinosinusitis and Head and Neck Cancers.

Inflammation is an etiological factor of various chronic diseases contributing to more than 50% of worldwide deaths. In this study, we focus on the immunosuppressive role of the programmed death-1 (PD-1) receptor and its ligand (PD-L1) in inflammatory-related diseases, including chronic rhinosinusitis and head and neck cancers. The study included 304 participants. Of this number, 162 patients had chronic rhinosinusitis with nasal polyps (CRSwNP), 40 patients had head and neck cancer (HNC) and there were 102 healthy subjects. The expression level of the PD-1 and PD-L1 genes in the tissues of the study groups was measured by qPCR and Western blot methods. The associations between the age of the patients and the extent of disease and genes' expression were evaluated. The study showed a significantly higher mRNA expression of PD-1 and PD-L1 in the tissues of both the CRSwNP and HNC patient groups compared to the healthy group. The severity of CRSwNP significantly correlated with the mRNA expression of PD-1 and PD-L1. Similarly, the age of the NHC patients influenced PD-L1 expression. In addition, a significantly higher level of PD-L1 protein was noticed also for both the CRSwNP and HNC patient groups. The increased expression of PD-1 and PD-L1 may be a potential biomarker of inflammatory-related diseases, including chronic rhinosinusitis and head and neck cancers.

Assessment of Subjective Tinnitus Treatment Results Using a Prototype Device for Electrical and Magnetic Stimulation of the Ear-Preliminary Study.

Background: The aim of the study was to evaluate the effectiveness of subjective tinnitus treatment in patients with cochlear sensorineural hearing loss with magnetic ear stimulation using a prototype device. Since the 1970s, studies have been conducted on the use of electrical stimulation of the ear in the treatment of tinnitus. The available literature contains various hypotheses about the influence of electrical stimulation of the ear on tinnitus. Material and Methods: Preclinical studies were performed for 100 patients, 40 women and 60 men (124 ears in total), aged 38-72 years, treated for tinnitus. A subjective assessment of the loudness of tinnitus was performed, and the frequency and intensity as well as hearing threshold were determined using a prototype device for electro-magnetic stimulation of the ear. The treatment cycle consisted of 10 five-minute stimulations performed daily 5 times a week. Results: Before treatment, persistent tinnitus was found in 100 ears (80.6%) and periodic tinnitus in 24 ears (19.4%). Immediately after treatment, persistent tinnitus was present only in 50 ears (40.3%) and periodic tinnitus in 40 ears (32.3%). Complete resolution of tinnitus was noted in 34 ears (27.4%). On the other hand, the examination performed 3 months after the treatment showed persistent tinnitus in 40 ears (32.3%) and periodic tinnitus in 50 ears (40.3%), and complete resolution of tinnitus was recorded in 34 ears (27.4%). Based on the VAS analog scale, there was an improvement in tinnitus in 98 ears (79.0%) immediately after treatment and no improvement in 26 ears (20.0%). The mean VAS scale before treatment was 4.9 points, after treatment it was 2.1 points and 3 months after treatment it was 1.9 points. Conclusions: The preliminary research results show the high effectiveness of magnetic stimulation in the treatment of tinnitus with the use of a prototype device for electromagnetic stimulation of the ear. There was no negative effect of the stimulation on hearing or tinnitus.

Expression of Programmed Death Receptor 1 (PD-1) Gene and Its Ligand (PD-L1) in Patients with Laryngeal Cancer.

(1) Background: The interaction of the programmed death receptor (PD-1) with its ligand 1 (PD-L1) allows cancer cells to escape from the control of the immune system. Research evaluating the expression of immune checkpoint genes in the tissues of laryngeal tumors may contribute to the introduction of new effective immunotherapeutic methods in this group of neoplasms. The aim of this study was to evaluate the expression of the gene for the programmed death receptor (PD-1) and its ligand (PD-L1) in laryngeal tumors (T1, T2, T3) in patients without lymph node involvement and distant metastases. (2) Methods: The study included 73 patients: 39 of them were diagnosed with carcinoma planoepiteliale keratodes (study group) and 34 with nasal septal deviation undergoing septoplasty (control group). Biological material for molecular tests (Real time PCR) was collected during surgical procedures. Furthermore, all study participants completed a questionnaire regarding, among others, smoking and body weight. (3) Results: Gene expression for programmed death receptor 1 (PD-1) and its ligand 1 (PD-L1) was, statistically, significantly higher (p < 0.0001) in tumor tissue than in unchanged mucosa. Moreover, it was found that the greater the tumor size, the higher the expression level of the tested molecules. (4) Conclusions: Although further research on the role of the PD-1/PD-L1 pathway in laryngeal tumors is necessary, the presented reports are promising and may constitute a contribution to considerations on the introduction of targeted immunotherapy with anti-PD1 and anti-PD-L1 monoclonal antibodies in the treatment of these tumors.

A survey of human histone H1 subtypes interaction networks: Implications for histones H1 functioning.

To show a spectrum of histone H1 subtypes (H1.1-H1.5) activity realized through the protein-protein interactions, data selected from APID resources were processed with sequence-based bioinformatics approaches. Histone H1 subtypes participate in over half a thousand interactions with nuclear and cytosolic proteins (ComPPI database) engaged in the enzymatic activity and binding of nucleic acids and proteins (SIFTER tool). Small-scale networks of H1 subtypes (STRING network) have similar topological parameters (P > .05) which are, however, different for networks hubs between subtype H1.1 and H1.4 and subtype H1.3 and H1.5 (P < .05) (Cytoscape software). Based on enriched GO terms (g:Profiler toolset) of interacting proteins, molecular function and biological process of networks hubs is related to RNA binding and ribosome biogenesis (subtype H1.1 and H1.4), cell cycle and cell division (subtype H1.3 and H1.5) and protein ubiquitination and degradation (subtype H1.2). The residue propensity (BIPSPI predictor) and secondary structures of interacting surfaces (GOR algorithm) as well as a value of equilibrium dissociation constant (ISLAND predictor) indicate that a type of H1 subtypes interactions is transient in term of the stability and medium-strong in relation to the strength of binding. Histone H1 subtypes bind interacting partners in the intrinsic disorder-dependent mode (FoldIndex, PrDOS predictor), according to the coupled folding and binding and mutual synergistic folding mechanism. These results evidence that multifunctional H1 subtypes operate via protein interactions in the networks of crucial cellular processes and, therefore, confirm a new histone H1 paradigm relating to its functioning in the protein-protein interaction networks.

Retrospective evaluation of risk factors for oral cavity and oropharynx cancers in patients under the program of head and neck cancers prevention.

INTRODUCTION: The aim of the study was to analyze the risk factors for oral cavity and pharyngeal cancer in people examined under the Head and Neck Cancer Prophylaxis Program. MATERIAL AND METHODS: The study was conducted in a total of 300 patients, including 186 women and 114 men, as part of the Head and Neck Cancer Prevention Program in 2014-2018. Before the laryngological examination, the patients completed a demographic and medical questionnaire regarding the risk factors of head and neck cancer, including education, reported disease symptoms, smoking addiction, number of cigarettes smoked daily, alcohol consumption, frequency of visits in a dental office, oral hygiene measures, number of sexual partners, oral sex, family medical history of head and neck cancer. RESULTS: The subjects reported the following symptoms: hoarseness 43.33%, difficulty swallowing 21.33%, pain or mouth burning 20.33% and other symptoms were observed in 46.33%. The main dental symptoms were: bleeding from the gums during teeth brushing in 48.89%, dry mouth 45.56%. Currently 20.33% of respondents smoke, whereas 54% of patients smoked in the past. In the analyzed material, the majority (80%) consumed alcoholic beverages. 27.67% of respondents admitted having oral sex, including 24.73% of women and 32.46% of men. After performing the extended diagnostics, the tumor was found in 10% of the subjects. C onclusions: Statistical significance of differences was found: between hoarseness and alcohol consumption, both in women and in men, between hoarseness and smoking in women, between difficulty in swallowing and smoking in women, between burning/pain in the mouth and smoking in men, between hoarseness and the cultivation of oral sex in men, between the difficulty of swallowing and the practice of oral sex in the studied men and between burning/pain in the oral cavity and the occurrence of malignancy.

[Narrow-band imaging endoscopy for diagnosis of adult laryngeal papillomatosis - case raports].

The laryngeal papillomas belong to the group of non-malignant tumours. The risk of getting sick increases with the number of contingent sexual contacts, smoking, alcohol abuse and untreated gastro-oesophageal reflux. This paper describes five cases presenting different levels of exposure to the risk factors and variable course of adult laryngeal papillomatosis. These people, in addition to routine diagnostics, were examined using endoscopy with the use of narrow beam of light, which turns out to be a useful diagnostic tool in the case of laryngeal papillomatosis.

Significance of avian linker histone (H1) polymorphic variation.

Most of avian histone H1 non-allelic subtypes, i.e. eight out of nine, show polymorphic heterogeneity manifested by the presence of two or three allelic variants formed as a result of amino acid deletion and substitution. In addition, some of histone H1 non-allelic subtypes exhibit various allelic complements in different bird species leading to the widening of a whole pool of histone H1 polymorphic variation. A wide range of histone H1 heterogeneity may indicate that the polymorphic variants can individually modulate some histone H1-dependent cellular processes by showing allele-specific influence on chromatin organization and function. Although, the exact way of avian histone H1 allelic variants' activity is not known, their structural separateness inferred from biochemical experiments and relationship with some characteristics of organism functioning disclosed in the genetic studies seem to confirm their importance. The aim of this review is to characterize the molecular origin of histone H1 polymorphisms and draw attention to the link between the histone H1 polymorphic variants and avian organismal features related to the physiological effects of bird individuals' living in the natural and breeding populations.

Brown Hare's (Lepus europaeus) Histone H1 Variant H1.2 as an Indicator of Anthropogenic Stress.

From the liver tissues of brown hare individuals that lived in two various habitats, i.e., the agricultural region with the predominant farms and the industrial area near a metallurgical plant, histones H1 were analyzed to compare their within and between population variability. Furthermore, because agricultural production emits mainly organic pollutants and metallurgical industry is a primarily source of inorganic contaminations, we wanted to check how the brown hare individuals are sensitive for both agents. Among brown hare H1 histones, the histone H1.2 was determined as heterogeneous due to its varied mobility in two-dimensional SDS-polyacrylamide gel. The obtained electrophoretic patterns contained differently moving single spots of histone H1.2 and also its double spots have a similar rate of electrophoretic mobility. Based on this, two homozygous phenotypes (slowly migrating 2a and faster moving 2b) and a heterozygous phenotype (2a2b) was distinguished. The relatively low variable (CV < 0.25) and comparably abundant (p > 0.05) histone H1.2 homozygous phenotypes form a heterozygous phenotype in a similar proportion, at a ratio approximating 0.5. Although the brown hare population originating from agricultural area displayed a slight excess of heterozygous individuals 2a2b (F = - 0.04), it was conformed to the Hardy-Weinberg assumption (χ2 = 0.035, p = 0.853). Compared with this population, a sevenfold reduced frequency of the phenotype 2b and above tenfold increase of a heterozygosity (F = - 0.53) was observed in the brown hare population inhabiting the vicinity of metallurgical plant. Therefore, this population did not fit to the Hardy-Weinberg law (χ2 = 5.65, p = 0.017). Despite the negligible genetic differentiation (FST = 0.026) between brown hare populations inhabiting areas with different anthropogenic pressure, a statistically significant difference in the distribution of their phenotypes (χ2 = 6.01, p = 0.049) and alleles (χ2 = 6.50, p = 0.013) was noted. The collected data confirm that the brown hare species is sensitive for environmental quality and may serve as a good indicator of habitat conditions related to both organic pollution emitted by agricultural activities (PIC = 0.48) and inorganic contamination originating from metallurgical processes (PIC = 0.49). These difference in the environmental quality might be assessed by estimation of genetic variability among the brown hare populations, based on the phenotypes distribution of histone H1 variant H1.2, the protein that was not so far employed as a molecular marker of anthropogenic stress.

A heterogeneity of the pheasant (Phasianus colchicus L.) erythrocyte histone H1 subtype H5.

In a previous work (Górnicka-Michalska et al. (1998)), an occurrence of genetic variants in the chicken erythrocyte histone H5 has been presented. Here, the pheasant histone H5 heterogeneity is characterized to verify if the interspecies variability of this protein is caused by the analogous determinants. During screening histone H1 preparations isolated from the pheasant erythrocytes, histone H5 was identified as differently located in the electrophoretic gels. According to the rate of electrophoretic migration, two histone H5 phenotypes (H5a and H5b) possessing similar quantitative proportion (P>0.05) were distinguished. A rare phenotype H5a (frequency 0.26) migrating faster in the SDS-PAGE was low mobile in the AU-PAGE, in contrast to the frequent phenotype H5b (frequency 0.74) that moved slowly in the SDS-PAGE and roamed faster in the AU-PAGE. The electrophoretic properties of histone H5 phenotypes may reflect disparities in their net charge and molecular weight. Peptide maps of histone H5 phenotypes, obtained by partial chemical cleavage (NBS) and limited enzymatic digestion (α-chymotrypsin), revealed their C-peptides possessing the same electrophoretic mobility and the N-peptides having variable rate of the electrophoretic migration. Based on this, the identified phenotypic variation seems to be determined by a histone H5 phenotype-specific amino acid sequence region situated in the N-terminal portion of its molecule. According to the identified varied sequence stretches, histone H5 phenotype may induce specific effects related to the organization and/or function of the pheasant chromatin.

Modulation of chromatin function through linker histone H1 variants.

In this review, the structural aspects of linker H1 histones are presented as a background for characterization of the factors influencing their function in animal and human chromatin. The action of H1 histone variants is largely determined by dynamic alterations of their intrinsically disordered tail domains, posttranslational modifications and allelic diversification. The interdependent effects of these factors can establish dynamic histone H1 states that may affect the organization and function of chromatin regions.

Nuclear and nucleolar activity of linker histone variant H1.0.

Histone H1.0 belongs to the class of linker histones (H1), although it is substantially distinct from other histone H1 family members. The differences can be observed in the chromosomal location and organization of the histone H1.0 encoding gene, as well as in the length and composition of its amino acid chain. Whereas somatic (H1.1-H1.5) histone H1 variants are synthesized in the cell cycle S-phase, histone H1.0 is synthesized throughout the cell cycle. By replacing somatic H1 variants during cell maturation, histone H1.0 is gradually deposited in low dividing cells and achieves the highest level of expression in the terminally differentiated cells. Compared to other differentiation-specific H1 histone (H5) characteristic for unique tissue and organisms, the distribution of histone H1.0 remains non-specific. Classic investigations emphasize that histone H1.0 is engaged in the organization of nuclear chromatin accounting for formation and maintenance of its nucleosomal and higher-order structure, and thus influences gene expression. However, the recent data confirmed histone H1.0 peculiar localization in the nucleolus and unexpectedly revealed its potential for regulation of nucleolar, RNA-dependent, activity via interaction with other proteins. According to such findings, histone H1.0 participates in the formation of gene-coded information through its control at both transcriptional and translational levels. In order to reappraise the biological significance of histone H1.0, both aspects of its activity are presented in this review.

Abundance of intrinsic structural disorder in the histone H1 subtypes.

The intrinsically disordered proteins consist of partially structured regions linked to the unstructured stretches, which consequently form the transient and dynamic conformational ensembles. They undergo disorder to order transition upon binding their partners. Intrinsic disorder is attributed to histones H1, perceived as assemblers of chromatin structure and the regulators of DNA and proteins activity. In this work, the comparison of intrinsic disorder abundance in the histone H1 subtypes was performed both by the analysis of their amino acid composition and by the prediction of disordered stretches, as well as by identifying molecular recognition features (MoRFs) and ANCHOR protein binding regions (APBR) that are responsible for recognition and binding. Both human and model organisms-animals, plants, fungi and protists-have H1 histone subtypes with the properties typical of disordered state. They possess a significantly higher content of hydrophilic and charged amino acid residues, arranged in the long regions, covering over half of the whole amino acid residues in chain. Almost complete disorder corresponds to histone H1 terminal domains, including MoRFs and ANCHOR. Those motifs were also identified in a more ordered histone H1 globular domain. Compared to the control (globular and fibrous) proteins, H1 histones demonstrate the increased folding rate and a higher proportion of low-complexity segments. The results of this work indicate that intrinsic disorder is an inherent structural property of histone H1 subtypes and it is essential for establishing a protein conformation which defines functional outcomes affecting on DNA- and/or partner protein-dependent cell processes.

Allelic isoforms of the chicken and duck histone H1.a.

Two isoforms of the erythrocyte histone H1.a were identified in two conservative flocks of Rhode Island Red chickens and six conservative flocks of ducks. The H1.a1 and H1.a2 isoforms formed three phenotypes (a1, a2 and a1a2) and were electrophoretically similar in the two species. The frequency of phenotype and histone H1.a allele occurrence varied within the genetic groups of birds, but the relatively rare allele a(2) was only detected in chicken and duck strains with colored feathers. Using mass spectrometry, we established that the difference between the measured masses of the duck H1.a isoforms was 156 Da. Since this value corresponds to the mass of the arginine residue alone or to the combined mass of the valine and glycine residues, we believe that the polymorphism of duck histone H1.a might have originated from sequence variation. A mass difference of 1 Da observed between chicken H1.a isoforms corresponded well to the previously detected Glu/Lys substitution (0.9414 Da) at position 117.

Linker histone subtypes and their allelic variants.

Members of histone H1 family bind to nucleosomal and linker DNA to assist in stabilization of higher-order chromatin structures. Moreover, histone H1 is involved in regulation of a variety of cellular processes by interactions with cytosolic and nuclear proteins. Histone H1, composed of a series of subtypes encoded by distinct genes, is usually differentially expressed in specialized cells and frequently non-randomly distributed in different chromatin regions. Moreover, a role of specific histone H1 subtype might be also modulated by post-translational modifications and/or presence of polymorphic isoforms. While the significance of covalently modified histone H1 subtypes has been partially recognized, much less is known about the importance of histone H1 polymorphic variants identified in various plant and animal species, and human cells as well. Recent progress in elucidating amino acid composition-dependent functioning and interactions of the histone H1 with a variety of molecular partners indicates a potential role of histone H1 polymorphic variation in adopting specific protein conformations essential for chromatin function. The histone H1 allelic variants might affect chromatin in order to modulate gene expression underlying some physiological traits and, therefore could modify the course of diverse histone H1-dependent biological processes. This review focuses on the histone H1 allelic variability, and biochemical and genetic aspects of linker histone allelic isoforms to emphasize their likely biological relevance.

Linker histone H1.b is polymorphic in grey partridge (Perdix perdix).

This study was aimed at characterizing allelic variations of erythrocyte histone H1.b by comparing the electrophoretic patterns of histone H1.b from individuals of grey partridge (Perdix perdix) population. As two alloforms, H1.b1 and H1.b2, were discerned in the screening gels, the histone H1.b was regarded to be a polymorphic protein encoded by a gene with two codominant alleles, b1 and b2, at a locus. The tested population was found to be at Hardy-Weinberg equilibrium (chi2 = 0.834, p = 0.361), with only a minor heterozygote deficiency (fixation index F = 0.136). Since the histone H1.b alloforms were identified in a two-dimensional gel containing sodium dodecyl sulfate, with no significant differences in their migration pattern in an one-dimensional acetic acid polyacrylamide gel, we assumed that the H1.b alloforms possessed a similar net charge and differed in their apparent molecular weights. A comparison of N-bromosuccinimide-cleaved and alpha-chymotrypsin-digested products of histone H1.b alloforms revealed slight differences in the velocity of C-terminal peptides and a similarity in migration of their N-terminal peptides in one-dimensional sodium dodecyl sulfate-polyacrylamide gel. Therefore, it seemed that the histone H1.b alloforms might differ in this amino acid sequence in a protein segment between N-bromosuccinimide cleavage site and the very C-terminus.

Chromatin compaction in terminally differentiated avian blood cells: the role of linker histone H5 and non-histone protein MENT.

Chromatin has a tendency to shift from a relatively decondensed (active) to condensed (inactive) state during cell differentiation due to interactions of specific architectural and/or regulatory proteins with DNA. A promotion of chromatin folding in terminally differentiated avian blood cells requires the presence of either histone H5 in erythrocytes or non-histone protein, myeloid and erythroid nuclear termination stage-specific protein (MENT), in white blood cells (lymphocytes and granulocytes). These highly abundant proteins assist in folding of nucleosome arrays and self-association of chromatin fibers into compacted chromatin structures. Here, we briefly review structural aspects and molecular mode of action by which these unrelated proteins can spread condensed chromatin to form inactivated regions in the genome.

Two polymorphic linker histone loci in Guinea fowl erythrocytes.

A variable migration of linker histone H1.b and H1.c spots in two-dimensional polyacrylamide gel patterns of total erythrocyte histone H1 has been detected during population screening in two differently plumaged Guinea fowl strains. Alloforms, H1.b1 and H1.b2 as well as H1.c1 and H1.c2, differing in apparent molecular weights tended to form only phenotypes b1 and b2 or c1 and c2 in a white-feathered strain while all phenotypes (b1, b2 and b1b2 or c1, c2 and c1c2, respectively) were present in a black-feathered population. Accordingly, the white-feathered population significantly deviated from the Hardy-Weinberg principle (chi-square test, d.f=1, p<<0.001) due to a lack of heterozygotes while the black-feathered population conformed to the Hardy-Weinberg equilibrium (p>0.05) at both H1.b and H1.c loci. Differential electrophoretic mobilities of the C-peptides from a partial chemical cleavage (N-bromosuccinimide) or limited enzymatic digestion (α-chymotrypsin and protease V8) of the histone H1.b and H1.c alloforms seem to indicate that altered amino acid sequence segments might be located either at the C-terminal end of globular domain or in the C-terminal domain itself.

Phenotypic variation of erythrocyte linker histone H1.c in a pheasant (Phasianus colchicus L.) population.

Our goal was to characterize a phenotypic variation of the pheasant erythrocyte linker histone subtype H1.c. By using two-dimensional polyacrylamide gel electrophoresis three histone H1.c phenotypes were identified. The differently migrating allelic variants H1.c1 and H1.c2 formed either two homozygous phenotypes, c1 and c2, or a single heterozygous phenotype, c1c2. In the pheasant population screened, birds with phenotype c2 were the most common (frequency 0.761) while individuals with phenotype c1 were rare (frequency 0.043).

Phenotypic variation of erythrocyte linker histone H1. c in a pheasant (Phasianus colchicus L.) population

Our goal was to characterize a phenotypic variation of the pheasant erythrocyte linker histone subtype H1.c. By using two-dimensional polyacrylamide gel electrophoresis three histone H1.c phenotypes were identified. The differently migrating allelic variants H1.c1 and H1.c2 formed either two homozygous phenotypes, c1 and c2, or a single heterozygous phenotype, c1c2. In the pheasant population screened, birds with phenotype c2 were the most common (frequency 0.761) while individuals with phenotype c1 were rare (frequency 0.043).