The enzymatic dissociation of human solid tissues is a critical process for disaggregating extracellular matrix and the isolation of individual cells for various applications, including the immortalizing primary cells, creating novel cell lines, and performing flow cytometry and its specialized type, FACS, as well as conducting scRNA-seq studies. Tissue dissociation procedures should yield intact, highly viable single cells that preserve morphology and cell surface markers. However, endocrine tissues, such as adrenal gland tumors, thyroid carcinomas, and pituitary neuroendocrine tumors, present unique challenges due to their complex tissue organization and morphological features. Our study conducted a morphological examination of these tissues, highlighting the intricate structures and secondary degenerative changes that complicate the dissociation process. We investigated the effects of various dissociation parameters, including the types of enzymes, incubation duration, and post-dissociation purification procedures, such as debris removal and nontarget blood cell lysis, on the viability of cells derived from different tumor types. The findings emphasize the importance of optimizing tissue digestion protocols to preserve cell viability and integrity, ensuring reliable outcomes for downstream analyses.

Current scRNA-seq studies of solid tissues mostly rely on enzymatic dissociation of fresh samples or the fallback on nuclei isolation from frozen or partially fixed samples. However, due to the complex tissue organization or cell fragility, it could be challenging to apply these approaches to the sensitive endocrine tissues. That is, dissociating intact cells from such problematic fresh-frozen samples routinely collected by biobanks remains challenging. In this study, we adapted the acetic-methanol dissociation method – ACME High Salt (ACME HS) to effectively isolate intact single cells from fresh-frozen endocrine tumor samples, including adrenal gland neoplasms, thyroid carcinomas, and pituitary neuroendocrine tumors. We compared the ability of enzymatic, ACME HS, and nuclear isolation methods to preserve the integrity of major cell types and gene expression across 41 tissue samples of different origins. We demonstrated that ACME HS simultaneously dissociates and fixes cells, thus preserving morphology and a high RNA integrity number in problematic cell types. This finding renders the ACME HS dissociation method a valuable alternative in scRNA-seq protocols for challenging tissues where obtaining live cell suspension is difficult or impossible.

Current dissociation methods for solid tissues in scRNA-seq studies do not guarantee intact single-cell isolation, especially for sensitive and complex human endocrine tissues. Most studies rely on enzymatic dissociation of fresh samples or nuclei isolation from frozen samples. Dissociating whole intact cells from fresh-frozen samples, commonly collected by biobanks, remains a challenge. Here, we utilized the acetic-methanol dissociation approach (ACME) to capture transcriptional profiles of individual cells from fresh-frozen tissue samples. This method combines acetic acid-based dissociation and methanol-based fixation. In our study, we optimized this approach for human endocrine tissue samples for the first time. We incorporated a high-salt washing buffer instead of the standard PBS to stabilize RNA and prevent RNases reactivation during rehydration. We have designated this optimized protocol as ACME HS (ACetic acid-MEthanol High Salt). This technique aims to preserve cell morphology and RNA integrity, minimizing transcriptome changes and providing a more accurate representation of mature mRNA. We compared the ability of enzymatic, ACME HS, and nuclei isolation methods to preserve major cell types, gene expression, and standard quality parameters across 41 tissue samples. Our results demonstrated that ACME HS effectively dissociates and fixes cells, preserving cell morphology and high RNA integrity. This makes ACME HS a valuable alternative for scRNA-seq protocols involving challenging tissues where obtaining a live cell suspension is difficult or disruptive.

The review covers modern diagnostic methods: Sanger sequencing and next-generation sequencing (NGS), polymerase chain reaction (PCR) and its modifications in real time (RT-PCR), enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC) and diagnostic test systems (DTS) developed on their basis. Using common chronic and infectious diseases as an example, the fundamental principles and modern versions of these methods are discussed, indicating their advantages and limitations for clinical use. The current problems and factors hindering the widespread introduction of these diagnostic methods into healthcare practice in Russia, as well as directions of their development in the near future, are analyzed.

The paper aims to define the genetic structure (strains, serotypes) of coronavirus SARS-CoV-2 and influenza viruses as well as assess the infection rate among the Moscow population and suggest measures for effective management of the epidemiological situation. Patients with upper respiratory tract infections (URTI) symptoms undergo naso-oropharyngeal swabbing procedure. The taken samples are used in polymerase chain reaction (PCR) testing and next generation sequencing (NGS) by means of MiSeq system (Illumina, US) for SARS-CoV-2 and influenza А and В viruses detection. The authors perform a bioinformatic analysis of genome sequences using Iterative Refinement Meta-Assembler (IRMA 1.2.1), Basic Local Alignment Search Tool (BLAST 6), and Phylogenetic Assignment of Named Global Outbreak Lineages (PANGOLIN). From late 2021 to early 2022, the genetic structure of COVID-19 diagnosed in Moscow residents changed. Thus, the sublineage BA of the Omicron variant replaced the sublineage AY of the SARS-CoV-2 Delta strain. Consequently, the infection rate rose to 3700 per 100,000 people. Associated with the appearance of less virulent Omicron sublineages (XBB, JN.1, FliRT, KS1, LR1, LF, FL1), the rate between the end of 2022 and the start of 2025 stood at 100–500 per 100,000 individuals. Between late 2022 and early 2023, 64.3–97.6% of the specified population was infected with the H1N1 strain of influenza A. However, since January 23, 2023, the B variant became prevalent, counting 52.36–97.37% of all test swabs in early 2023. From the end of 2023 to the beginning of 2024, Moscow citizens contracted 2 novel H3N2 strains of influenza A: A/Massachusetts/01/2020 and A/Massachusetts/38/2019. From late 2024 to early 2025, Moscow residents were diagnosed with a newly emerged subclone of influenza A strain, i.e. H1N1, Hawaii/25/2020, and the Texas/43/2019 variant of influenza B. The outcomes of this research hold the potential to advance the modernization of PCR-based diagnostic kits, aid in the selection of vaccine strain candidates, and evaluate the effectiveness of established vaccines and antiviral treatments.

The adult adrenal cortex undergoes constant renewal, yet underlying human-specific mechanisms remain poorly understood. Here we generated single-cell and spatial transcriptomic atlases of adult human and mouse adrenal glands, leveraging single-cell-resolution spatial data and a rare clonal mosaic case for lineage inference. In humans, we identified age-associated zona glomerulosa (ZG) cell states with direct cortisol synthesis capacity and sex-specific differences in inferred cholesterol balance. Cross-species comparison revealed conserved aldosterone-producing ZG but notable divergence in zona fasciculata markers, absence of zona reticularis homologs in mice and differential SHH–WNT4 signaling in proliferating cells. We uncovered human WT1 ⁻ capsule-to-ZG transition and vascular smooth muscle cell-to-steroidogenic transitions supported by mosaic lineage evidence. We revealed dispersed proliferating cortical SF1 ⁺ EZH2 ⁺ cells throughout the human cortex in contrast with ZG restriction in mice. Taken together, our data expand the centripetal renewal model and establish a comparative framework for human adrenocortical biology.