Composition of the sputum bacterial microbiome of patients with different pathomorphological forms of non-small-cell lung cancer.

Recent studies have shown that the bacterial microbiome of the respiratory tract influences the development of lung cancer. Changes in the composition of the microbiome are observed in patients with chronic inflammatory processes. Such microbiome changes may include the occurrence of bacteria that cause oxidative stress and that are capable of causing genome damage in the cells of the host organism directly and indirectly. To date, the composition of the respiratory microbiome in patients with various histological variants of lung cancer has not been studied. In the present study, we determined the taxonomic composition of the sputum microbiome of 52 patients with squamous cell carcinoma of the lung, 52 patients with lung adenocarcinoma and 52 healthy control donors, using next-generation sequencing (NGS) on the V3-V4 region of the bacterial gene encoding 16S rRNA. The sputum microbiomes of patients with different histological types of lung cancer and controls did not show significant differences in terms of the species richness index (Shannon); however, the patients differed from the controls in terms of evenness index (Pielou). The structures of bacterial communities (beta diversity) in the adenocarcinoma and squamous cell carcinoma groups were also similar; however, when analyzed according to the matrix constructed by the Bray-Curtis method, there were differences between patients with squamous cell carcinoma and healthy subjects, but not between those with adenocarcinoma and controls. Using the LEFse method it was possible to identify an increase in the content of Bacillota (Streptococcus and Bacillus) and Actinomycetota (Rothia) in the sputum of patients with squamous cell carcinoma when compared with samples from patients with adenocarcinoma. There were no differences in the content of bacteria between the samples of patients with adenocarcinoma and the control ones. The content of representatives of the genera Streptococcus, Bacillus, Peptostreptococcus (phylum Bacillota), Prevotella, Macellibacteroides (phylum Bacteroidota), Rothia (phylum Actinomycetota) and Actinobacillus (phylum Pseudomonadota) was increased in the microbiome of sputum samples from patients with squamous cell carcinoma, compared with the control. Thus, the sputum bacterial microbiome of patients with different histological types of non-small-cell lung cancer has significant differences. Further research should be devoted to the search for microbiome biomarkers of lung cancer at the level of bacterial species using whole-genome sequencing.

Genetic damage in lymphocytes of lung cancer patients is correlated to the composition of the respiratory tract microbiome.

Recent findings indicate that the microbiome may have significant impact on the development of lung cancer by its effects on inflammation, dysbiosis or genome damage. The aim of this study was to compare the sputum microbiome of lung cancer (LC) patients with the chromosomal aberration (CA) and micronuclei (MN) frequency in peripheral blood lymphocytes. In the study, the taxonomic composition of the sputum microbiome of 66 men with untreated LC were compared with 62 control subjects with respect to CA and MN frequency and centromere fluorescence in situ hybridisation analysis. Results showed a significant increase in CA (4.11 ± 2.48% versus 2.08 ± 1.18%) and MN (1.53 ± 0.67% versus 0.87 ± 0.49%) frequencies, respectively, in LC patients as compared to control subjects. The higher frequency of centromeric positive MN of LC patients was mainly due to aneuploidy. A significant increase in Streptococcus, Bacillus, Gemella and Haemophilus in LC patients was detected, in comparison to the control subjects while 18 bacterial genera were significantly reduced, which indicates a decrease in the beta diversity in the microbiome of LC patients. Although, the CA frequency in LC patients is significantly associated with an increased presence of the genera Bacteroides, Lachnoanaerobaculum, Porphyromonas, Mycoplasma and Fusobacterium in their sputum, and a decrease for the genus Granulicatella after application of false discovery rate correction, significance was not any more present. The decrease of MN frequency of LC patients is significantly associated with an increase in Megasphaera genera and Selenomonas bovis. In conclusion, a significant difference in beta diversity of microbiome between LC and control subjects and association between the sputum microbiome composition and genome damage of LC patients was detected, thus supporting previous studies suggesting an etiological connection between the airway microbiome and LC.

Taxonomic diversity of sputum microbiome in lung cancer patients and its relationship with chromosomal aberrations in blood lymphocytes.

Here we report a pilot-sized study to compare the taxonomic composition of sputum microbiome in 17 newly-diagnosed lung cancer (LC) patients and 17 controls. Another object was to compare the representation of individual bacterial genera and species in sputum with the frequency of chromosomal aberrations in the blood lymphocytes of LC patients and in controls. Both groups were male; average age 56.1 ± 11.5 in patients and 55.7 ± 4.1 in controls. Differences in the species composition of bacterial communities in LC patients and controls were significant (pseudo-F = 1.94; p = 0.005). Increased prevalence in LC patients was detected for the genera Haemophilus and Bergeyella; whereas a decrease was observed for the genera Atopobium, Stomatobaculum, Treponema and Porphyromonas. Donors with high frequencies of chromosomal aberrations had a significant reduction in the microbiome of representatives of the genus Atopobium in the microbiome and a simultaneous increase in representatives of the species Alloprevotella compared to donors with a low level of chromosomal aberrations in lymphocytes. Thus, a comparison of the bacterial composition in the sputum of donors with cytogenetic damages in theirs lymphocytes, warrants further investigations on the potential role of microorganisms in the process of mutagenesis in somatic cells of the host body.

Induction and modulation of genotoxicity by the bacteriome in mammals.

The living environment is a multilevel physical and chemical xenobiotic complex with potentially mutagenic effects and health risks. In addition to inorganic exposures, all terrestrial and aquatic living forms interact with microbiota as selectively established communities of bacteria, viruses and fungi. Along these lines, the human organism should then be considered a "meta-organism" with complex dynamics of interaction between the environment and microbiome. Bacterial communities within the microbiome, bacteriome, by its mass, symbiotic or competitive position and composition are in a fragile balance with the host organisms and have a crucial impact on their homeostasis. Bacteriome taxonomic composition is modulated by age, sex and host genetic profile and may be changed by adverse environmental exposures and life style factors such as diet or drug intake. A changed and/or misbalanced bacteriome has genotoxic potential with significant impact on the pathogenesis of acute, chronic and neoplastic diseases in the host organism. Bacteria may produce genotoxins, express a variety of pathways in which they generate free radicals or affect DNA repair causing genome damage, cell cycle arrest and apoptosis, modulate immune response and launch carcinogenesis in the host organism. Future investigations should focus on the interplay between exposure to xenobiotics and bacteriome composition, immunomodulation caused by misbalanced bacteriome, impact of the environment on bacteriome composition in children and its lifelong effect on health risks.

SYK interaction with ITGß4 suppressed by Epstein-Barr virus LMP2A modulates migration and invasion of nasopharyngeal carcinoma cells.

Epstein-Barr virus (EBV)-encoded Latent Membrane Protein 2A (LMP2A) is an EBV latency-associated protein regularly expressed in nasopharyngeal carcinoma (NPC). In B cells, LMP2A activity resembles that of a constitutively activated antigen receptor, which recruits the Syk tyrosine kinase to activate a set of downstream signaling pathways. LMP2A also downregulates cellular Syk levels. In the present study, we demonstrate that Syk interacts with the integrin ß4 subunit (ITGß4) of integrin α6ß4 in epithelial cells and that concurrent LMP2A expression interferes with this interaction by competitive binding to Syk. We find that both Syk and LMP2A have an effect on ITGß4 cell surface expression. However, in LMP2A expressing cells, ITGß4 remains concentrated at the cellular protrusions, an expression pattern characteristic of motile cells, including NPC-derived epithelial cells. This effect of LMP2A on ITGß4 localization is associated with a greater propensity for migration and invasion in-vitro, and may contribute to the invasive property of LMP2A-expressing NPC.

Stem cell gene expression in MRPS18-2-immortalized rat embryonic fibroblasts.

We have recently found that primary rat embryonic fibroblasts (REFs) could be immortalized by overexpression of the human mitochondrial ribosomal protein MRPS18-2 (S18-2). A derived cell line, designated 18IM, expressed the embryonic stem cell markers SSEA-1 and Sox2. Upon inoculation into severe combined immunodeficiency mice, 18IM cells differentiated to express pan-keratin. They were not tumorigenic. Here we report the gene profiling of 18IM, compared with REF cells. Pathways involved in oxidative phosphorylation, ubiquinone (Coenzyme Q 10) biosynthesis, fatty acid elongation in mitochondria, PI3K/AKT signaling, a characteristic of rapidly proliferating cells, were upregulated in 18IM. Genes involved in the transcription/translation machinery and redox reactions, like elongation factors, ATP synthases, NADH dehydrogenases, mitogen activated kinases were upregulated as well. 18IM cells produced more pyruvate, indicating enhanced ATP synthesis. The expression of Oct4, Sox2, and Nanog that can contribute to the experimental induction of pluripotency in primary fibroblasts was also elevated, in contrast to Klf4 and C-myc that were downregulated. Subsequently, three new immortalized cell lines were produced by S18-2 overexpression in order to check the representativeness of 18IM. All of them showed anchorage-independent growth pattern. Two of three clones lost vimentin and smooth muscle actin, and expressed Sox2 and Oct4. We suggest that S18-2 is involved in the developmental regulation.

The Shb signalling scaffold binds to and regulates constitutive signals from the Epstein-Barr virus LMP2A membrane protein.

The Epstein-Barr virus latency-associated membrane protein LMP2A has been shown to activate the survival kinase Akt in epithelial and B cells in a phosphoinositide 3-kinase-dependent fashion. In this study, we demonstrate that the signalling scaffold Shb associates through SH2 and PTB domain interactions with phosphorylated tyrosine motifs in the LMP2A N-terminal tail. Additionally, we show that mutation of tyrosines in these motifs as well as shRNA-mediated downregulation of Shb leads to a loss of constitutive Akt-activation in LMP2A-expressing cells. Furthermore, utilization by Shb of the LMP2A ITAM motif regulates stability of the Syk tyrosine kinase in LMP2A-expressing cells. Our data set the precedent for viral utilization of the Shb signalling scaffold and implicate Shb as a regulator of LMP2A-dependent Akt activation.

C-terminal domain of the Epstein-Barr virus LMP2A membrane protein contains a clustering signal.

The latency-regulated transmembrane protein LMP2A interferes with signaling from the B-cell antigen receptor by recruiting the tyrosine kinases Lyn and Syk and by targeting them for degradation by binding the cellular E3 ubiquitin ligase AIP4. It has been hypothesized that this constitutive activity of LMP2A requires clustering in the membrane, but molecular evidence for this has been lacking. In the present study we show that LMP2A coclusters with chimeric rat CD2 transmembrane molecules carrying the 27-amino-acid (aa) intracellular C terminus of LMP2A and that this C-terminal domain fused to the glutathione-S-transferase protein associates with LMP2A in cell lysates. This molecular association requires neither the cysteine-rich region between aa 471 and 480 nor the terminal three aa 495 to 497. We also show that the juxtamembrane cysteine repeats in the LMP2A C terminus are the major targets for palmitoylation but that this acylation is not required for targeting of LMP2A to detergent-insoluble glycolipid-enriched membrane microdomains.

Latent membrane protein 2A of Epstein-Barr virus binds WW domain E3 protein-ubiquitin ligases that ubiquitinate B-cell tyrosine kinases.

The latent membrane protein (LMP) 2A of Epstein-Barr virus (EBV) is implicated in the maintenance of viral latency and appears to function in part by inhibiting B-cell receptor (BCR) signaling. The N-terminal cytoplasmic region of LMP2A has multiple tyrosine residues that upon phosphorylation bind the SH2 domains of the Syk tyrosine kinase and the Src family kinase Lyn. The LMP2A N-terminal region also has two conserved PPPPY motifs. Here we show that the PPPPY motifs of LMP2A bind multiple WW domains of E3 protein-ubiquitin ligases of the Nedd4 family, including AIP4 and KIAA0439, and demonstrate that AIP4 and KIAA0439 form physiological complexes with LMP2A in EBV-positive B cells. In addition to a C2 domain and four WW domains, these proteins have a C-terminal Hect catalytic domain implicated in the ubiquitination of target proteins. LMP2A enhances Lyn and Syk ubiquitination in vivo in a fashion that depends on the activity of Nedd4 family members and correlates with destabilization of the Lyn tyrosine kinase. These results suggest that LMP2A serves as a molecular scaffold to recruit both B-cell tyrosine kinases and C2/WW/Hect domain E3 protein-ubiquitin ligases. This may promote Lyn and Syk ubiquitination in a fashion that contributes to a block in B-cell signaling. LMP2A may potentiate a normal mechanism by which Nedd4 family E3 enzymes regulate B-cell signaling.

A new picornavirus isolated from bank voles (Clethrionomys glareolus).

A previously unknown picornavirus was isolated from bank voles (Clethrionomys glareolus). Electron microscopy images and sequence data of the prototype isolate, named Ljungan virus, showed that it is a picornavirus. The amino acid sequences of predicted Ljungan virus capsid proteins VP2 and VP3 were closely related to the human pathogen echovirus 22 (approximately 70% similarity). A partial 5' noncoding region sequence of Ljungan virus showed the highest degree of relatedness to cardioviruses. Two additional isolates were serologically and molecularly related to the prototype.

[Use of collase for culturing cells]. / Primenenie kollazy dlia kul'tivirovaniia kletok.

Collase, an enzymatic preparation made from crab hepatopancreas, was used as a dissociative reagent in preparation of primary cell cultures and for detachment of continuous cells from the substrate during reinoculation. Collase was found to increase viable cell harvest by 2 to 2.5 times in comparison with trypsin and had a less detrimental effect on the cells which retained their proliferative activity, morphology, productivity, and sensitivity to viruses.

[Identification of the gene for the immunodominant p35 protein from vaccinia virus]. / Identifikatsiia gena immunodominantnogo belka p35 virusa ospovaktsiny.

A major immunodominant envelope protein p35 of vaccina virus was purified by means of extraction from virions with detergent NP-40. The protein was cleaved with CNBr, four homogenous peptides were isolated and their N-terminal amino acid sequences were determined. Computer search in a protein sequences data bank revealed that the immunodominant protein p35 of vaccinia virus is encoded by H3 gene in HindIII-H fragment of vaccinia virus genome.