Gut microbiome encoded purine and amino acid pathways present prospective biomarkers for predicting metformin therapy efficacy in newly diagnosed T2D patients.

Metformin is widely used for treating type 2 diabetes mellitus (T2D). However, the efficacy of metformin monotherapy is highly variable within the human population. Understanding the potential indirect or synergistic effects of metformin on gut microbiota composition and encoded functions could potentially offer new insights into predicting treatment efficacy and designing more personalized treatments in the future. We combined targeted metabolomics and metagenomic profiling of gut microbiomes in newly diagnosed T2D patients before and after metformin therapy to identify potential pre-treatment biomarkers and functional signatures for metformin efficacy and induced changes in metformin therapy responders. Our sequencing data were largely corroborated by our metabolic profiling and identified that pre-treatment enrichment of gut microbial functions encoding purine degradation and glutamate biosynthesis was associated with good therapy response. Furthermore, we identified changes in glutamine-associated amino acid (arginine, ornithine, putrescine) metabolism that characterize differences in metformin efficacy before and after the therapy. Moreover, metformin Responders' microbiota displayed a shifted balance between bacterial lipidA synthesis and degradation as well as alterations in glutamate-dependent metabolism of N-acetyl-galactosamine and its derivatives (e.g. CMP-pseudaminate) which suggest potential modulation of bacterial cell walls and human gut barrier, thus mediating changes in microbiome composition. Together, our data suggest that glutamine and associated amino acid metabolism as well as purine degradation products may potentially condition metformin activity via its multiple effects on microbiome functional composition and therefore serve as important biomarkers for predicting metformin efficacy.

Gut Microbiome Composition and Dynamics in Hospitalized COVID-19 Patients and Patients with Post-Acute COVID-19 Syndrome.

The gut microbiome plays a pivotal role in the modulation of host responses during viral infections, and recent studies have underscored its significance in the context of coronavirus disease 2019 (COVID-19). We aimed to investigate the dynamics and compositional changes in the gut microbiome of COVID-19 patients, addressing both the acute phase and the recovery process, with a particular focus on the emergence of post-COVID-19 conditions. Involving 146 COVID-19 patients and 110 healthy controls, this study employed a shotgun metagenomics approach for cross-sectional and longitudinal analyses with one- and three-month follow-ups. We observed a decline in taxonomic diversity among hospitalized COVID-19 patients compared to healthy controls, while a subsequent increase in alpha diversity was shown during the recovery process. A notable contribution of Enterococcus faecium was identified in the acute phase of the infection, accompanied by an increasing abundance of butyrate-producing bacteria (e.g., Roseburia, Lachnospiraceae_unclassified) during the recovery period. We highlighted a protective role of the Prevotella genus in the long-term recovery process and suggested a potential significance of population-specificity in the early gut microbiome markers of post-acute COVID-19 syndrome. Our study represents distinctive gut microbiome signatures in COVID-19, with potential diagnostic and prognostic implications, pinpointing potential modulators of the disease progression.

Prevalence of SARS-CoV-2 in domestic cats (Felis catus) during COVID-19 pandemic in Latvia.

BACKGROUND: The causative agent of the COVID-19 pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is of zoonotic origin and has shown reverse zoonotic transmissibility. OBJECTIVES: The aim of this cross-sectional study was to investigate the serological and molecular prevalence of SARS-CoV-2 infection in the domestic cat (Felis catus) population from Latvia in natural conditions and subsequently perform viral genome analysis. METHODS: Oropharyngeal and rectal swabs and blood samples were collected from 273 domestic cats during the second wave of COVID-19 infection in Latvia. Molecular prevalence was determined by using reverse transcriptase-polymerase chain reaction (RT-PCR). Serum samples were analysed via double antigen enzyme-linked immunosorbent assay targeting the antibody against the nucleocapsid protein of SARS-CoV-2. Positive swab samples were analysed using whole viral genome sequencing and subsequent phylogenetic analysis of the whole genome sequencing data of the samples was performed. RESULTS: The overall SARS-CoV-2 RT-PCR positivity and seroprevalence was 1.1% (3/273) and 2.6% (7/273), respectively. The SARS-CoV-2 genome sequences from three RT-PCR positive cats were assigned to the three common lineages (PANGOLIN lineage S.1.; B.1.177.60. and B.1.1.7.) circulating in Latvia during the particular period of time. CONCLUSIONS: These findings indicate that feline infection with SARS-CoV-2 occurred during the second wave of the COVID-19 pandemic in Latvia, yet the overall prevalence was low. In addition, it seems like no special 'cat' pre-adaptations were necessary for successful infection of cats by the common lineages of SARS-CoV-2.

Whole-Genome Sequencing of 502 Individuals from Latvia: The First Step towards a Population-Specific Reference of Genetic Variation.

Despite rapid improvements in the accessibility of whole-genome sequencing (WGS), understanding the extent of human genetic variation is limited by the scarce availability of genome sequences from underrepresented populations. Developing the population-scale reference database of Latvian genetic variation may fill the gap in European genomes and improve human genomics research. In this study, we analysed a high-coverage WGS dataset comprising 502 individuals selected from the Genome Database of the Latvian Population. An assessment of variant type, location in the genome, function, medical relevance, and novelty was performed, and a population-specific imputation reference panel (IRP) was developed. We identified more than 18.2 million variants in total, of which 3.3% so far are not represented in gnomAD and dbSNP databases. Moreover, we observed a notable though distinct clustering of the Latvian cohort within the European subpopulations. Finally, our findings demonstrate the improved performance of imputation of variants using the Latvian population-specific reference panel in the Latvian population compared to established IRPs. In summary, our study provides the first WGS data for a regional reference genome that will serve as a resource for the development of precision medicine and complement the global genome dataset, improving the understanding of human genetic variation.

Abundance and prevalence of ESBL coding genes in patients undergoing first line eradication therapy for Helicobacter pylori.

The spread of extended-spectrum beta-lactamases (ESBLs) in nosocomial and community-acquired enterobacteria is an important challenge for clinicians due to the limited therapeutic options for infections that are caused by these organisms. Here, we developed a panel of ESBL coding genes, evaluated the abundance and prevalence of ESBL encoding genes in patients undergoing H. pylori eradication therapy, and summarized the effects of eradication therapy on functional profiles of the gut microbiome. To assess the repertoire of known beta lactamase (BL) genes, they were divided into clusters according to their evolutionary relation. Primers were designed for amplification of cluster marker regions, and the efficiency of this amplification panel was assessed in 120 fecal samples acquired from 60 patients undergoing H. pylori eradication therapy. In addition, fecal samples from an additional 30 patients were used to validate the detection efficiency of the developed ESBL panel. The presence for majority of targeted clusters was confirmed by NGS of amplification products. Metagenomic sequencing revealed that the abundance of ESBL genes within the pool of microorganisms was very low. The global relative abundances of the ESBL-coding gene clusters did not differ significantly among treatment states. However, at the level of each cluster, classical ESBL producers such as Klebsiella sp. for blaOXY (p = 0.0076), Acinetobacter sp. for blaADC (p = 0.02297) and others, differed significantly with a tendency to decrease compared to the pre- and post-eradication states. Only 13 clusters were common across all three datasets, suggesting a patient-specific distribution profile of ESBL-coding genes. The number of AMR genes detected in the post-eradication state was higher than that in the pre-eradication state, which could be attributed, at least in part, to the therapy. This study demonstrated that the ESBL screening panel was effective in targeting ESBL-coding gene clusters from bacterial DNA and that minor differences exist in the abundance and prevalence of ESBL-coding gene levels before and after eradication therapy.

Metatranscriptome analysis of blood in healthy individuals and irritable bowel syndrome patients.

Introduction. Although the presence of micro-organisms in the blood of healthy humans is a relatively new concept, there is a growing amount of evidence that blood might have its own microbiome.Gap Statement. Previous research has targeted the taxonomic composition of the blood microbiome using DNA-based sequencing methods, while little information is known about the presence of microbial transcripts obtained from the blood and their relation to conditions connected with increased gut permeability.Aim. To detect potentially alive and active micro-organisms and investigate differences in taxonomic composition between healthy people and patients with irritable bowel syndrome (IBS), we used the metatranscriptomics approach.Methodology. We collected blood samples from 23 IBS patients and 26 volunteers from the general population, and performed RNAseq on the isolated RNA. Reads corresponding to microbial genomes were identified with Kraken 2's standard plus protozoa and fungi database, and re-estimated at genus level with Bracken 2.7. We looked for trends in the taxonomic composition, making a comparison between the IBS and control groups, accounting for other different factors.Results. The dominant genera in the blood microbiome were found to be Cutibacterium, Bradyrhizobium, Escherichia, Pseudomonas, Micrococcus, Delftia, Mediterraneibacter, Staphylococcus, Stutzerimonas and Ralstonia. Some of these are typical environmental bacteria and could partially represent contamination. However, analysis of sequences from the negative controls suggested that some genera which are characteristic of the gut microbiome (Mediterraneibacter, Blautia, Collinsella, Klebsiella, Coprococcus, Dysosmobacter, Anaerostipes, Faecalibacterium, Dorea, Simiaoa, Bifidobacterium, Alistipes, Prevotella, Ruminococcus) are less likely to be a result of contamination. Differential analysis of microbes between groups showed that some taxa associated with the gut microbiome (Blautia, Faecalibacterium, Dorea, Bifidobacterium, Clostridium, Christensenella) are more prevalent in IBS patients compared to the general population. No significant correlations with any other factors were identified.Conclusion. Our findings support the existence of the blood microbiome and suggest the gut and possibly the oral microbiome as its origin, while the skin microbiome is a possible but less certain source. The blood microbiome is likely influenced by states of increased gut permeability such as IBS.

Transcriptome of GH-producing pituitary neuroendocrine tumours and models are significantly affected by somatostatin analogues.

Pituitary neuroendocrine tumours (PitNETs) are neoplasms of the pituitary that overproduce hormones or cause unspecific symptoms due to mass effect. Growth hormone overproducing GH-producing PitNETs cause acromegaly leading to connective tissue, metabolic or oncologic disorders. The medical treatment of acromegaly is somatostatin analogues (SSA) in specific cases combined with dopamine agonists (DA), but almost half of patients display partial or full SSA resistance and potential causes of this are unknown. In this study we investigated transcriptomic landscape of GH-producing PitNETs on several levels and functional models-tumour tissue of patients with and without SSA preoperative treatment, tumour derived pituispheres and GH3 cell line incubated with SSA to study effect of medication on gene expression. MGI sequencing platform was used to sequence total RNA from PitNET tissue, pituispheres, mesenchymal stromal stem-like cells (MSC), and GH3 cell cultures, and data were analysed with Salmon-DeSeq2 pipeline. We observed that the GH-producing PitNETs have distinct changes in growth hormone related pathways related to its functional status alongside inner cell signalling, ion transport, cell adhesion and extracellular matrix characteristic patterns. In pituispheres model, treatment regimens (octreotide and cabergoline) affect specific cell proliferation (MKI67) and core functionality pathways (RYR2, COL8A2, HLA-G, ARFGAP1, TGFBR2). In GH3 cells we observed that medication did not have transcriptomic effects similar to preoperative treatment in PitNET tissue or pituisphere model. This study highlights the importance of correct model system selection for cell transcriptomic profiling and data interpretation that could be achieved in future by incorporating NGS methods and detailed cell omics profiling in PitNET model research.

Genome wide analysis of circulating miRNAs in growth hormone secreting pituitary neuroendocrine tumor patients' plasma.

Background: Circulating plasma miRNAs have been increasingly studied in the field of pituitary neuroendocrine tumor (PitNET) research. Our aim was to discover circulating plasma miRNAs species associated with growth hormone (GH) secreting PitNETs versus assess how the plasma levels of discovered miRNA candidates are impacted by SSA therapy and whether there is a difference in their levels between GH secreting PitNETs versus other PitNET types and healthy individuals. Design: We compared plasma miRNA content and levels before and after surgery focusing on GH secreting PitNET patients. Selected miRNA candidates from our data and literature were then tested in a longitudinal manner in somatostatin analogues (SSA) treatment group. Additionally, we validated selected targets in an independent GH secreting PitNET group. Methods: miRNA candidates were discovered using the whole miRNA sequencing approach and differential expression analysis. Selected miRNAs were then analyzed using real-time polymerase chain reaction (qPCR). Results: Whole miRNA sequencing discovered a total of 16 differentially expressed miRNAs (DEMs) in GH secreting PitNET patients' plasma 24 hours after surgery and 19 DEMs between GH secreting PitNET patients' plasma and non-functioning (NF) PitNET patients' plasma. Seven miRNAs were selected for further testing of which miR-625-5p, miR-503-5p miR-181a-2-3p and miR-130b-3p showed a significant downregulation in plasma after 1 month of SSA treatment. mir-625-5p was found to be significantly downregulated in plasma of GH secreting PitNET patients vs. NF PitNET patients. miR-625-5p alongside miR-130b-3p were also found to be downregulated in GH PitNETs compared to healthy individuals. Conclusions: Our study suggests that expression of plasma miRNAs miR-625-5p, miR-503-5p miR-181a-2-3p and miR-130b-3p in GH secreting PitNETs is affected by SSA treatment. Additionally, miR-625-5p can distinguish GH secreting PitNETs from other PitNET types and healthy controls warranting further research on these miRNAs for treatment efficacy.

Whole exome sequencing reveals novel risk genes of pituitary neuroendocrine tumors.

Somatic genetic alterations in pituitary neuroendocrine tumors (PitNET) tissues have been identified in several studies, but detection of overlapping somatic PitNET candidate genes is rare. We sequenced and by employing multiple data analysis methods studied the exomes of 15 PitNET patients to improve discovery of novel factors involved in PitNET development. PitNET patients were recruited to the study before PitNET removal surgery. For each patient, two samples for DNA extraction were acquired: venous blood and PitNET tissue. Exome sequencing was performed using Illumina NexSeq 500 sequencer and data analyzed using two separate workflows and variant calling algorithms: GATK and Strelka2. A combination of two data analysis pipelines discovered 144 PitNET specific somatic variants (mean = 9.6, range 0-19 per PitNET) of which all were SNVs. Also, we detected previously known GNAS PitNET mutation and identified somatic variants in 11 genes, which have contained somatic variants in previous WES and WGS studies of PitNETs. Noteworthy, this is the third study detecting somatic variants in gene RYR1 in the exomes of PitNETs. In conclusion, we have identified two novel PitNET candidate genes (AC002519.6 and AHNAK) with recurrent somatic variants in our PitNET cohort and found 13 genes overlapping from previous PitNET studies that contain somatic variants. Our study demonstrated that the use of multiple sequencing data analysis pipelines can provide more accurate identification of somatic variants in PitNETs.

Role of Somatostatin Signalling in Neuroendocrine Tumours.

Somatostatin (SST) is a small peptide that exerts inhibitory effects on a wide range of neuroendocrine cells. Due to the fact that somatostatin regulates cell growth and hormone secretion, somatostatin receptors (SSTRs) have become valuable targets for the treatment of different types of neuroendocrine tumours (NETs). NETs are a heterogeneous group of tumours that can develop in various parts of the body, including the digestive system, lungs, and pituitary. NETs are usually slow growing, but they are often diagnosed in advanced stages and can display aggressive behaviour. The mortality rate of NETs is not outstandingly increased compared to other malignant tumours, even in the metastatic setting. One of the intrinsic properties of NETs is the expression of SSTRs that serve as drug targets for SST analogues (SSAs), which can delay tumour progression and downregulate hormone overproduction. Additionally, in many NETs, it has been demonstrated that the SSTR expression level provides a prognostic value in predicting a therapeutic response. Furthermore, higher a SSTR expression correlates with a better survival rate in NET patients. In recent studies, other epigenetic regulators affecting SST signalling or SSA-mTOR inhibitor combination therapy in NETs have been considered as novel strategies for tumour control. In conclusion, SST signalling is a relevant regulator of NET functionality. Alongside classical SSA treatment regimens, future advanced therapies and treatment modalities are expected to improve the disease outcomes and overall health of NET patients.

Dominance of Fructose-Associated Fructobacillus in the Gut Microbiome of Bumblebees (Bombus terrestris) Inhabiting Natural Forest Meadows.

Bumblebees are key pollinators in agricultural landscapes. However, little is known about how gut microbial communities respond to anthropogenic changes. We used commercially produced colonies of buff-tailed bumblebees (Bombus terrestris) placed in three habitats. Whole guts (midgut, hindgut, and rectum) of B. terrestris specimens were dissected from the body and analyzed using 16S phylogenetic community analysis. We observed significantly different bacterial community composition between the agricultural landscapes (apple orchards and oilseed rape (Brassica napus) fields) and forest meadows, whereas differences in gut communities between the orchards and oilseed rape fields were nonsignificant. Bee-specific bacterial genera such as Lactobacillus, Snodgrassella, and Gilliamella dominated gut communities of B. terrestris specimens. In contrast, the guts of B. terrestris from forest meadows were dominated by fructose-associated Fructobacillus spp. Bacterial communities of workers were the most diverse. At the same time, those of males and young queens were less diverse, possibly reflecting greater exposure to the colony's inner environment compared to the environment outside the colony, as well as bumblebee age. Our results suggest that habitat quality, exposure to environmental microbes, nectar quality and accessibility, and land use significantly affect gut bacterial composition in B. terrestris.

First Report on the Latvian SARS-CoV-2 Isolate Genetic Diversity.

Remaining a major healthcare concern with nearly 29 million confirmed cases worldwide at the time of writing, novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused more than 920 thousand deaths since its outbreak in China, December 2019. First case of a person testing positive for SARS-CoV-2 infection within the territory of the Republic of Latvia was registered on 2nd of March 2020, 9 days prior to the pandemic declaration by WHO. Since then, more than 277,000 tests were carried out confirming a total of 1,464 cases of coronavirus disease 2019 (COVID-19) in the country as of 12th of September 2020. Rapidly reacting to the spread of the infection, an ongoing sequencing campaign was started mid-March in collaboration with the local testing laboratories, with an ultimate goal in sequencing as much local viral isolates as possible, resulting in first full-length SARS-CoV-2 isolate genome sequences from the Baltics region being made publicly available in early April. With 133 viral isolates representing ~9.1% of the total COVID-19 cases during the "first coronavirus wave" in the country (early March, 2020-mid-September, 2020) being completely sequenced as of today, here, we provide a first report on the genetic diversity of Latvian SARS-CoV-2 isolates.

Pituispheres Contain Genetic Variants Characteristic to Pituitary Adenoma Tumor Tissue.

The most common type of pituitary neoplasms is benign pituitary adenoma (PA). Clinically significant PAs affect around 0.1% of the population. Currently, there is no established human PA cell culture available and when PA tumor cells are cultured they form two distinct types depending on culturing conditions either free-floating aggregates also known as pituispheres or cells adhering to the surface of cell plates and displaying mesenchymal stem-like properties. The aim of this study was to trace the origin of sphere-forming and adherent pituitary cell cultures and characterize the potential use of these surgery derived cell lines as PA model. We carried out a paired-end exome sequencing of patients' tumor and germline DNA using Illumina NextSeq followed by characterization of corresponding PA cell cultures. Variation analysis revealed a low amount of somatic mutations (mean = 5.2, range 3-7) in exomes of PAs. Somatic mutations of the primary surgery material can be detected in the exomes of respective pituispheres, but not in exomes of respective mesenchymal stem-like cells. For the first time, we show that the genome of pituispheres represents genome of PA while mesenchymal stem cells derived from the PA tissue do not contain mutations characteristic to PA in their genome, therefore, most likely representing normal cells of pituitary or surrounding tissues. This finding indicates that pituispheres can be used as a human model of PA cells, but combination of cell culturing techniques and NGS needs to be employed to adjust for disability to propagate spheres in culturing conditions.

Selective enrichment of heterotrophic nitrifiers Alcaligenaceae and Alcanivorax spp. from industrial wastewaters.

Removal of nitrogen from wastewaters (WW) represents a global problem. The low nitrification rate during WW treatment is often caused by ecotoxicity. This problem is attributed mostly to the industrial WW. Our study was focused on the testing of industrial WW and activated sludge (AS) with the aim to reveal the abundance of nitrifiers and increase their biomass, thus, providing the additional step, i.e., bioaugmentation, within the technological process of WW treatment. Plating of AS on the selective solidified media designated for the 1st and 2nd nitrification stages, resulted in the shift in bacterial community structure with dominated Alcaligenaceae and Alcanivorax for the 1st stage, and Alcanivorax-for the 2nd stage of nitrification, respectively. Incubation of AS in the presence of real WW and selective nitrification broth resulted in a considerable increase (one or two magnitudes in the presence of the 1st and 2nd stage nitrification broth, respectively) of culturable nitrifiers after 5 days incubation under aerated conditions. The obtained data provide with evidence about a possibility to strengthen the role of heterotrophic nitrifiers in the treatment of industrial WW, where toxicity obstacles inhibited nitrification under conventional conditions.

Case report: recurrent pituitary adenoma has increased load of somatic variants.

BACKGROUND: Pituitary adenomas (PA) have an increased potential for relapse in one to 5 years after resection. In this study, we investigated the genetic differences in genomic DNA of primary and rapidly recurrent tumours in the same patient to explain the causality mechanisms of PA recurrence. CASE PRESENTATION: The patient was a 69-year-old female with non-functional pituitary macroadenoma with extension into the left cavernous sinus (Knosp grade 2) who underwent craniotomy and partial resection in August 2010. Two years later, the patient had prolonged tumour growth with an essential suprasellar extension (Knosp grade 2), and a second craniotomy with partial tumour resection was performed in September 2012. In both tumours, the KI-67 level was below 1.5%. Exome sequencing via semiconductor sequencing of patient germline DNA and somatic DNA from both tumours was performed. Tmap alignment and Platypus variant calling were performed followed by variant filtering and manual review with IGV software. We observed an increased load of missense variants in the recurrent PA tumour when compared to the original tumour. The number of detected variants increased from ten to 26 and potential clonal expansion of four variants was observed. Additionally, targeted SNP analysis revealed five rare missense SNPs with a potential impact on the function of the encoded proteins. CONCLUSIONS: In this case study, an SNP located in HRAS is the most likely candidate inducing rapid PA progression. The relapsed PA tumour had a higher variation load and fast tumour recurrence in this patient could be caused by clonal expansion of the leftover tumour tissue.

Medication for Acromegaly Reduces Expression of MUC16, MACC1 and GRHL2 in Pituitary Neuroendocrine Tumour Tissue.

Acromegaly is a disease mainly caused by pituitary neuroendocrine tumor (PitNET) overproducing growth hormone. First-line medication for this condition is the use of somatostatin analogs (SSAs), that decrease tumor mass and induce antiproliferative effects on PitNET cells. Dopamine agonists (DAs) can also be used if SSA treatment is not effective. This study aimed to determine differences in transcriptome signatures induced by SSA/DA therapy in PitNET tissue. We selected tumor tissue from twelve patients with somatotropinomas, with half of the patients receiving SSA/DA treatment before surgery and the other half treatment naive. Transcriptome sequencing was then carried out to identify differentially expressed genes (DEGs) and their protein-protein interactions, using pathway analyses. We found 34 upregulated and six downregulated DEGs in patients with SSA/DA treatment. Three tumor development promoting factors MUC16, MACC1, and GRHL2, were significantly downregulated in therapy administered PitNET tissue; this finding was supported by functional studies in GH3 cells. Protein-protein interactions and pathway analyses revealed extracellular matrix involvement in the antiproliferative effects of this type of the drug treatment, with pronounced alterations in collagen regulation. Here, we have demonstrated that somatotropinomas can be distinguished based on their transcriptional profiles following SSA/DA therapy, and SSA/DA treatment does indeed cause changes in gene expression. Treatment with SSA/DA significantly downregulated several factors involved in tumorigenesis, including MUC16, MACC1, and GRHL2. Genes that were upregulated, however, did not have a direct influence on antiproliferative function in the PitNET cells. These findings suggested that SSA/DA treatment acted in a tumor suppressive manner and furthermore, collagen related interactions and pathways were enriched, implicating extracellular matrix involvement in this anti-tumor effect of drug treatment.

Evaluation of the Possibility to Detect Circulating Tumor DNA From Pituitary Adenoma.

Objective: Circulating free DNA (cfDNA) in general and circulating tumor DNA (ctDNA) in particular is becoming an increasingly used form of liquid biopsy biomarkers. In this study, we are investigating the ability to detect ctDNA from the plasma of pituitary adenoma (PA) patients. Design: Tumor tissue samples were obtained from planed PA resections, before which blood plasma samples were taken. Somatic variants found in PA tissue samples were evaluated in related cfDNA, isolated from plasma samples. Methods: Sanger sequencing, as well as previously obtained whole-exome sequencing data, were used to evaluate somatic variants composition in tumor tissue samples. cfDNA was isolated from the same PA patients and competitive allele-specific TaqMan PCR and amplicon-based next-generation sequencing (NGS) approach were used for targeted detection of variants found in corresponding tumor tissue samples. Results: Using NGS-based analysis, we detected five out of 17 somatic variants in 40 to 60% of total reads, three variants in 0.50-5.00% of total read count, including GNAS c.601C>T, which was detected using ultra-deep NGS (1.78 million X) in 0.77% of amplicons reads. Nine variants were not detected. We also detected We were not able to detect variant found in PA tissue in cfDNA using cast-PCR, indicating that the portion of variant-containing ctDNA in total isolated cfDNA is too small to be detected with this method. Conclusions: For the first time, we demonstrate the possibility to detect somatic variants of PA in cfDNA isolated from patients' blood plasma. Whether the source of variant detected in cfDNA is PA should be further tested.

Functional Characteristics of Multipotent Mesenchymal Stromal Cells from Pituitary Adenomas.

Pituitary adenomas are one of the most common endocrine and intracranial neoplasms. Although they are theoretically monoclonal in origin, several studies have shown that they contain different multipotent cell types that are thought to play an important role in tumor initiation, maintenance, and recurrence after therapy. In the present study, we isolated and characterized cell populations from seven pituitary somatotroph, nonhormonal, and lactotroph adenomas. The obtained cells showed characteristics of multipotent mesenchymal stromal cells as observed by cell morphology, cell surface marker CD90, CD105, CD44, and vimentin expression, as well as differentiation to osteogenic and adipogenic lineages. They are capable of growth and passaging under standard laboratory cell culture conditions and do not manifest any hormonal cell characteristics. Multipotent mesenchymal stromal cells are present in pituitary adenomas regardless of their clinical manifestation and show no considerable expression of somatostatin 1-5 and dopamine 2 receptors. Most likely obtained cells are a part of tissue-supportive cells in pituitary adenoma microenvironment.