DNA methylation meta-analysis confirms the division of the genome into two functional groups

Based on a meta-analysis of human genome methylation data, we tested a theoretical model in which aging is explained by the redistribution of limited resources in cells between two main tasks of the organism: its self-sustenance based on the function of the housekeeping gene group (HG) and functional differentiation, provided by the (IntG) integrative gene group. A meta-analysis of methylation of 100 genes, 50 in the HG group and 50 in IntG, showed significant differences ( p<0.0001) between our groups in the level of absolute methylation values of genes bodies and its promoters. We showed a reliable decrease of absolute methylation values in IntG with rising age in contrast to HG, where this level remained constant. The one-sided decrease in methylation in the IntG group is indirectly confirmed by the dispersion data analysis, which also decreased in the genes of this group. The imbalance between HG and IntG in methylation levels suggests that this IntG-shift is a side effect of the ontogenesis grownup program and the main cause of aging. The theoretical model of functional genome division also suggests the leading role of slow dividing and post mitotic cells in triggering and implementing the aging process.

Pseudomonas Tumuqiensis Sp. Nov., Isolated From Greenhouse Soil

A Gram-stain-negative, aerobic, rod-shaped and motile bacterium, named LAMW06T, was isolated from greenhouse soil in Beijing, China. In the 16S rRNA gene sequence comparison, strain LAMW06T had the highest similarity with Pseudomonas cuatrocienegasensis 1NT was 97.4%. Phylogenetic analysis base on the 16S rRNA and three housekeeping gene sequences (gyrB, rpoB and rpoD) indicated that LAMW06T represented a member of the genus Pseudomonas. The genome sequence size of the isolate was 5.5 Mb, with the DNA G+C content of 63.5 mol%. The average nucleotide identity and DNA–DNA hybridization values between strain LAMW06T and closely related members of Pseudomonas borbori R-20821T, Pseudomonas taeanensis MS-3T and Pseudomonas cuatrocienegasensis 1NT were 90.9%, 82.4%, 81.5 % and 43.0%, 25.9%, 24.6 % respectively. The major fatty acids contained summed feature 3 (C16:1 ω6c and/or C16:1 ω7c), C18:1 ω7c and C16:0. The primary respiratory quinone was ubiquinone-9. The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, six aminophospholipids, six phospholipids, one aminolipid and one glycolipid. According to the genotypic, phylogenetic and chemotaxonomic data, strain LAMW06T represents a novel species within the genus Pseudomonas, for which the name Pseudomonas tumuqiensis sp. nov. is proposed. The type strain is LAMW06T (=GDMCC 1.2003T =KCTC 72829T).

Identification of the best housekeeping gene for RT-qPCR analysis of human pancreatic organoids

In the last few years, there has been a considerable increase in the use of organoids, which is a new three-dimensional culture technology applied in scientific research. The main reasons for their extensive use are their plasticity and multiple applications, including in regenerative medicine and the screening of new drugs. The aim of this study was to better understand these structures by focusing on the choice of the best housekeeping gene (HKG) to perform accurate molecular analysis on such a heterogeneous system. This feature should not be underestimated because the inappropriate use of a HKG can lead to misleading data and incorrect results, especially when the subject of the study is innovative and not totally explored like organoids. We focused our attention on the newly described human pancreatic organoids (hPOs) and compared 12 well-known HKGs (ACTB, B2M, EF1α, GAPDH, GUSB, HPRT, PPIA, RNA18S, RPL13A TBP, UBC and YWHAZ). Four different statistical algorithms (NormFinder, geNorm, BestKeeper and ΔCt) were applied to estimate the expression stability of each HKG, and RefFinder was used to identify the most suitable genes for RT-qPCR data normalization. Our results showed that the intragroup and intergroup comparisons could influence the best choice of the HKG, making clear that the identification of a stable reference gene for accurate and reproducible RT-qPCR data normalization remains a critical issue. In summary, this is the first report on HKGs in human organoids, and this work provides a strong basis to pave the way for further gene analysis in hPOs.

Analysis of the Expression and Prognostic Value of MSH2 in Pan-Cancer Based on Bioinformatics

Background. MutS homolog 2 (MSH2), with the function of identifying mismatches and participating in DNA repair, is the “housekeeping gene” in the mismatch repair (MMR) system. MSH2 deficiency has been reported to enhance cancer susceptibility for the association of hereditary nonpolyposis colorectal cancer. However, the expression and prognostic significance of MSH2 have not been studied from the perspective of pan-cancer. Methods. The GTEx database was used to analyze the expression of MSH2 in normal tissues. The TCGA database was used to analyze the differential expression of MSH2 in pan-cancers. The prognostic value of MSH2 in pan-cancer was assessed using Cox regression and Kaplan-Meier analysis. Spearman correlations were used to measure the relationship between the expression level of MSH2 in pan-cancer and the level of immune infiltration, tumor mutational burden (TMB), and microsatellite instability (MSI). Results. MSH2 is highly expressed in most type of cancers and significantly correlated with prognosis. In COAD, KIRC, LIHC, and SKCM, the expression of MSH2 was significantly positively correlated with the abundance of B cells, CD4+ T cells, CD8+ T cells, dendritic cells, macrophages, and neutrophils. In THCA, MSH2 expression correlated with CD8+T Cell showed a significant negative correlation. MSH2 had significantly negative correlations with stromal score and immune score in a variety of cancers and significantly correlated with TMB and MSI of a variety of tumors. Conclusions. MSH2 may play an important role in the occurrence, development, and immune infiltration of cancer. MSH2 can emerge as a potential biomarker for cancer diagnosis and prognosis.

Identification of Trueperella bernardiae isolated from peking ducks (Anas platyrhynchos domesticus) by phenotypical and genotypical investigations and by a newly developed loop-mediated isothermal amplification (LAMP) assay

Trueperella (T.) bernardiae is a well-known bacterial pathogen in infections of humans, rarely in animals. In the present study, five T. bernardiae isolates, isolated from five Peking ducks of four different farms, were identified by phenotypic properties, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, and genotypically by sequencing the 16S ribosomal RNA (rRNA) gene, the superoxide dismutase A encoding gene sodA, and the glyceraldehyde-3-phosphate dehydrogenase encoding gene gap. In addition, the T. bernardiae isolates could be identified with a newly developed loop-mediated isothermal amplification (LAMP) assay based on the gyrase encoding housekeeping gene gyrA. All these tests clearly identified the T. bernardiae isolates to the species level. However, the detection of the specific gene gyrA with the newly designed LAMP assay appeared with a high sensitivity and specificity, and could help to identify this bacterial species in human and animal infections in future. The importance of the T. bernardiae isolates for the clinical condition of the ducks and for the problems at farm level remains unclear.

A Quadruplex qPCR for Detection and Differentiation of Classic and Natural Recombinant Myxoma Virus Strains of Leporids

A natural recombinant myxoma virus (referred to as ha-MYXV or MYXV-Tol08/18) emerged in the Iberian hare (Lepus granatensis) and the European rabbit (Oryctolagus cuniculus) in late 2018 and mid-2020, respectively. This new virus is genetically distinct from classic myxoma virus (MYXV) strains that caused myxomatosis in rabbits until then, by acquiring an additional 2.8 Kbp insert within the m009L gene that disrupted it into ORFs m009L-a and m009L-b. To distinguish ha-MYXV from classic MYXV strains, we developed a robust qPCR multiplex technique that combines the amplification of the m000.5L/R duplicated gene, conserved in all myxoma virus strains including ha-MYXV, with the amplification of two other genes targeted by the real-time PCR systems designed during this study, specific either for classic MYXV or ha-MYXV strains. The first system targets the boundaries between ORFs m009L-a and m009L-b, only contiguous in classic strains, while the second amplifies a fragment within gene m060L, only present in recombinant MYXV strains. All amplification reactions were validated and normalized by a fourth PCR system directed to a housekeeping gene (18S rRNA) conserved in eukaryotic organisms, including hares and rabbits. The multiplex PCR (mPCR) technique described here was optimized for Taqman® and Evagreen® systems allowing the detection of as few as nine copies of viral DNA in the sample with an efficiency > 93%. This real-time multiplex is the first fast method available for the differential diagnosis between classic and recombinant MYXV strains, also allowing the detection of co-infections. The system proves to be an essential and effective tool for monitoring the geographical spread of ha-MYXV in the hare and wild rabbit populations, supporting the management of both species in the field.

Detection of CCND1 Overexpression By RNA-Seq from Tna Samples As a Surrogate for t(11:14) Translocation Traditionally Measured By FISH in Multiple Myeloma Patients for Improved Patient Care

Background: Multiple myeloma (MM) is a blood cancer type affecting plasma cell in bone marrow. MM is heterogenous in nature but t(11;14)(q13;q32) translocation is a common prognostic marker among MM patients. One of the most frequent oncogenic drivers involved in this chromosomal rearrangement is CCND1 (Cyclin D1) gene translocation downstream to the immunoglobulin heavy chain (IGH), which results on overexpression of CCND1, thus promoting abnormal cell proliferation. Oncogenic CCND1 RNA levels can result from translocations such as t(11;14), gene amplifications, increased transcription rates and/or RNA stability. Indeed, CCND1 RNA overexpression has a favorable prognostic value for patients treated with high doses of chemotherapies but important challenges remain in accurate detection of CCND1 RNA levels. Currently, FISH is the gold standard method for detecting t(11:14) translocations at the DNA level. However, it cannot detect CCND1 overexpression. Therefore, a method that can detect CCND1 overexpression levels, as well as in frame transcripts has clinical implications. In the current study we leveraged in-use NeoGenomics Heme TNA single tube NGS assay to enable the detection of CCND1 RNA overexpression as a complementary test to FISH testing. Methods: We performed RNA sequencing from 32 healthy donors and on fixed cell pellets from 94 CD138-enriched BM samples from MM patients and from using the amplicon based (Qiagen, inc) NGS assay. We developed pipeline for gene expression by TPM count (transcript per million) for CCND1, and further normalized to the "housekeeping" gene GUSB. We validated the normalization to GUSB by comparing to normalization using the geometric mean of four housekeeping genes (GUSB, PGD, RPL5 and RPL19) showing a high correlation (R 2&gt;0.95). A commercially available qRT-PCR assay was used as orthogonal method to further confirm the linearity of the quantitative gene expression signal in NGS. The analytical cutoff was determined from normalized TPM calculation from 32 healthy volunteers following CLSI guideline (CLSI_EP17-A2) and further updated from MM-PCE samples with t(11:14) translocations from a CLIS-validated FISH assay . Results: From 94 CD138-enriched BM samples, 26 had t(11:14) translocations, or CCND1 gains as detected by FISH, 15 samples were confirmed negative by FISH and 32 normal volunteers with no suspected disease. Also, we determined the analytical cutoff for CCND1 overexpression based on the CLSI guidelines to be 2.37 times the expression level of GUSB ("housekeeping" gene) using normal volunteers (n=32) (sensitivity 86% and specificity 77%). We found specificity to be low, so further evaluated the threshold using a ROC curve analysis with multiple tests. Using Fischer's exact test, we found CCND1 expression 3.27 times the GUSB expression to yield higher specificity of 86.5 % and sensitivity for 78.9%. Further, we used 26 FISH positive and 32 normal samples to build a new model and determined the cutoff for CCND1 overexpression to be 4.15 times GUSB expression, which resulted lower sensitivity but higher specificity (75% sensitivity and 100% specificity). When we evaluated 15 FISH negative samples with this cutoff we observed CCND1 was not overexpressed in six samples, but 9 samples did have some degree of overexpression. Overexpression was confirmed by qRT-PCR. Two CCND1 high- and low- expressing normal samples (MM-PCE 27 and 48) were further evaluated using alternative extraction methods to test the dependencies on extractions and the data showed concordant to each other for overexpression. Interestingly, 1 sample (MM-PCE-27) showed very high overexpression without t(11:14) translocation event (~100 fold over expressed). Cytogenetic studies were discordant with FISH as well for this sample, showing abnormalities related to chr7q,13q,12p but no indication of any chr11 related event. Conclusions: In this study we evaluated our existing NGS assay for CCND1 overexpression using TNA as a surrogate for traditional FISH, while demonstrating the accuracy of the RNA quantitation by NGS using qRT-PCR. We developed an RNA-seq based CCND1 expression assay that could be used to complement traditional FISH testing especially if there is limited specimen. The confirmation of overexpression in FISH negative samples may suggest new ways to improve MM patients risk stratification and treatment. Disclosures Ghosal: NeoGenomics Laboratories: Current Employment. Alarcon: NeoGenomics Laboratories: Current Employment. Koo: Neo Genomics Laboratories: Current Employment. Kang: Neo Genomics Laboratories: Current Employment. Ramesh: Neo Genomics Laboratories: Current Employment. Gyuris: Neo Genomics Laboratories: Current Employment. Jung: NeoGenomics Laboratories, Inc.: Current Employment. Thomas: NeoGenomics Laboratories, Inc.: Current Employment. Fabunan: NeoGenomics Laboratories, Inc.: Current Employment. Magnan: NeoGenomics Laboratories, Inc.: Current Employment. Nam: NeoGenomics Laboratories, Inc.: Current Employment. Petersen: Neo Genomics Laboratories: Current Employment. Lopez-Diaz: NeoGenomics Laboratories, Inc.: Current Employment. Yamahata: Neo Genomics Laboratories: Current Employment. Bender: NeoGenomics Laboratories, Inc.: Current Employment. Agersborg: NeoGenomics Laboratories, Inc.: Current Employment. Ye: Neo Genomics Laboratories: Current Employment. Funari: NeoGenomics Laboratories, Inc.: Current Employment.

Comparative Analysis of Molecular-Genetic Properties in Non-Toxigenic Vibrio cholerae O1 Strains Biovar El Tor, Isolated in Russia and on Cholera Endemic Territories

Objective of the study was to perform a comparative analysis of molecular-genetic properties in non-toxigenic Vibrio cholerae O1 strains biovar El Tor, isolated in the Republic of Kalmykia and on cholera endemic territories and to reveal their phylogenetic relations to toxigenic isolates.Materials and methods. We have carried out bio-information analysis of whole genome sequences of 60 cholera vibrio strains from endemic as regards cholera regions and from Kalmykia. The presence of pathogenicity and endemicity islands in their genomes has been determined. Specifed have been the sequence-types of the examined strains and whole genome SNP-analysis conducted.Results and discussion. Non-toxigenic El Tor vibrios circulating in Kalmykia are clustered into two major genotypes: ctxA–tcpA+VPI-2+VSP– and ctxA–tcpA–VPI-2Δ+VSP–, where VPI-2 Δ+ signifes the presence of deletions of varying length in the genome of this pathogenicity island. Only the latter one is found in regions endemic for cholera. In addition, ctxA– tcpA+VPI-2+VSP+ populations circulate in cholera endemic foci, not found in Kalmykia. 17 sequence-types were identifed among the studied strains (by seven housekeeping gene loci). Phylogenetic analysis performed using SNP-typing demonstrated the absence of close genetic relation between the ctxA–tcpA+VPI-2+VSP– vibrios from Kalmykia and both toxigenic and non-toxigenic vibrios with different composition of pathogenicity and pandemicity islands in the genome. At the same time, genetic proximity of ctxA–tcpA–VPI-2Δ+VSP– cholera vibrios from endemic cholera foci with those isolated in Kalmykia was detected, which may indicate the possibility of their recurrent importation into the territory of Russia. Non-toxigenic V. cholerae strains found in the territory of Kalmykia are characterized by a high genetic diversity. Circulation of the strains with unique sequence-types suggests their potential for long-term persistence on this territory. At the same time, phylogenetic closeness and identity of certain strains with strains from endemic territories can be an evidence of repeated importation.

New Reference Genes for qRT-PCR Analysis as a Potential Target for Identification of Trichophyton verrucosum in Different Culture Conditions

Dermatophytes are a group of filamentous fungi infecting skin, hair, and nails that raise great diagnostic difficulties. qRT-PCR is a reliable technique for quantifying gene expression with increasingly frequent use in mycological diagnostics. Knowledge of genes and molecular markers with potential to be used in the identification of dermatophytes is of great importance for the development of this branch of diagnostics. In this article, the suitability of six candidate reference genes (TUBB, ACTB, ADPRF, RPL2, SDHA, and EEF1A1) was investigated for gene expression analysis in the dermatophyte Trichophyton verrucosum, which was cultured in various mycological media that are commonly used in a diagnostic laboratory, i.e., Sabouraud, potato dextrose, and keratin-supplemented MM-Cove. The different culture conditions are extremely important factors for the growth and physiology of dermatophytes. Gene expression stability was evaluated using geNorm, NormFinder, BestKeeper, and RefFinder algorithms. Regarding the stability of expression, SDHA was the most stable housekeeping gene; hence, this gene is recommended for future qRT-PCR studies on T. verrucosum strains. These results allow us to conclude that the SDHA gene can be an additional good candidate as an identification target in the qRT-PCR technique.