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The purpose is to study the genetic and agrotechnological features of the formation of the sowing qualities of oat grain at various agricultural backgrounds in the forest-steppe of the Trans-Urals. Materials and methods. The research was carried out on an experimental field located in the forest-steppe zone of the Trans-Urals. The object of the study were three varieties of oats, which were sown on different agricultural grounds, ensuring the formation of yields from 3.0 to 6.0 t/ha of grain. The mass of 1000 grains, the proportion of fine grains, germination energy and laboratory germination were determined. Results. On the natural agricultural background, the Talisman and Otrada varieties form a yield of 1.41 and 1.85 t/ha with a fraction of fine grain (< 2.0 mm) up to 11 %. In the Foma variety on the natural agricultural background, this indicator was 7.5 %. The introduction of mineral fertilizers for the planned yield from 3.0 to 5.0 t/ha reduces the amount of fine grain only in the Talisman and Foma varieties. Grain with high sowing qualities is formed on a natural agricultural background and when fertilizers are applied to the planned yield of 3.0 t/ha. The grain obtained on such variants had 72–76 % germination energy and 91-93% laboratory germination. Grain obtained at a high and very high agricultural background had low sowing indicators: germination energy and laboratory germination reached minimum values: 57–59 and 61–63 %, respectively. It was found that the mass of 1000 grains depends on fertilizers by 23.4 % and 30.0 % on the weather conditions of the growing season. Varietal characteristics account for 9.6 %. Laboratory germination of the studied varieties by 86.8 % depends on the level of mineral nutrition. Scientific novelty. For the first time for the Northern Trans-Urals, an optimal level of agricultural background has been established, providing maximum yield of oat grain with high sowing qualities. Recommendations. In the forest-steppe zone of the Trans-Urals, the cultivation of oats for seed purposes is recommended in fields with an average or increased agricultural background, designed to obtain yields of 3.0 and 4.0 t/ha.

Purpose. To assess the drought resistance of modern varieties of oats at the initial stage of ontogenesis by laboratory method. Methods. The following varieties of oats have been studied: Foma, Otrada, Talisman, Marshal, Ekspress, Assol’, Tigrovy and Petrovich. Relative drought resistance was determined by simulating drought by increasing the osmotic pressure with different concentrations of sucrose: 1.4; 4.4; 7.4; 10.5 and 16.6 %. This corresponded to osmotic pressures of 3, 6, 9, 12, and 18 atm. Distilled water was used as a control. The response of varieties to drought was determined by seed germination and depression of growth processes. Results. It was found that the varieties Talisman and Petrovich are characterized by very low resistance to drought – at an osmotic pressure of 3 atm, their germination was 29 and 52%, respectively. With a further increase in pressure, only single germinations with a very high degree of depression (more than 80%) were noted. Oat variety Ekspress was characterized by maximum resistance to drought. Its seeds germinated when the osmotic pressure increased to 12 atm (sucrose concentration reached 10.5 %). To a small extent, the Foma variety was inferior to it, which germinated worse than Ekspress, but had an average degree of depression of growth processes at an osmotic pressure of 3 to 12 atm. The rest of the studied oat varieties were moderately drought-resistant both in terms of germination and depression. Scientific novelty. The conducted analysis of variance showed that the relative drought resistance by 72 % depends on the concentration of sucrose, which indicates the inefficiency of the assessment by one concentration. The share of influence of a variety on genetically determined drought resistance is 10.3 %. This confirms the hypothesis that drought resistance is determined not only by genetic inheritance. It is recommended to use varieties Foma and Ekspress as parental forms in breeding for drought resistance.

The article discusses the main requirements for digital competencies of graduates of higher educational institutions for the oil and gas geological industry. A review of the main IT trends in the scientific and production activities of an oil and gas enterprise and a survey of the personnel skills necessary for their implementation is carried out, the requirements of an employer hiring a modern IT specialist are formulated and formalized.

The article discusses the results of a scientific project developing new methodological and technological approaches to the creation of geoinformation models for the purposes of waste and secondary resource management. The urgency and originality of the work are revealed, the goals and objectives of a geoinformation system, integrating a set of spatial information on the objects of the industry and on the key aspects of its development, are formulated. A description of the prototype of such system, implemented on the example of a pilot territory — the Arctic zone of the Russian Federation, — is given.

The article discusses the technology of forming a mapping database on objects involved in the field of waste management (sources of waste generation and recyclables, their processing enterprises and facilities for the disposal of industrial and household waste). Basic information resources providing information about such objects are analyzed, requirements for the database structure and description of technological stages of its formation are given. The proposed technology was successfully tested as part of the development of a prototype of a geographical information system for waste and secondary resources management, implemented on the example of the Arctic zone of the Russian Federation, as well as the creation of a geo-portal of the geographical information systems laboratory of Dubna State University, which provides geo-information support for the educational and research activities of the Institute for System Analysis and Management in terms of developing and maintaining cartographic databases.

The purpose of this work is to study the nature of inheritance of avenin components and description of new blocks of components in oat varieties cultivated in the Tyumen region for further use in marker breeding. Methods. We analyzed the grains of F2 hybrids from crosses of oat varieties ♀Tyumenskiy golozernyy × ♂Megion, ♀Tyumenskiy golozernyy × ♂Otrada, ♀Otrada × ♂Tyumenskiy golozernyy, ♀Megion × ♂Otrada. Native electrophoresis was performed in 13.2 % PAGE for 4.0–4.5 h at a constant voltage of 500 V. Results. In the course of research, it was found that the analyzed components of avenin make up blocks. The frequency distribution of phenotypic classes for each locus in each of the studied hybrid combinations corresponds to the assumption that each of the blocks is inherited as a single codominant mendelian unit. The synthesis of blocks of avenin components is controlled by three independent avenin-coding loci. Analysis of the inheritance of individual components of avenin in F2 hybrids made it possible to describe three new blocks of components, the synthesis of which is controlled by the Avn A locus – A9, A10, and A11; one block controlled by the Avn B locus – B7 and one block controlled by the Avn C locus – C8. On the basis of the results obtained, complete protein formulas were drawn up for the studied varieties of sowing oats: Tyumenskiy golozernyy – Avn 2.7.3; Megion – Avn 2 + 9.7.5; Otrada – Avn 10 + 11.4.8. It was found that the use of a spectrum of only one type as a standard is insufficient and leads to errors in the identification of component blocks. For more accurate identification, several varieties should be used simultaneously to obtain a complex spectrum-standard that combines several blocks of components for each locus. Scientific novelty. Described 5 new blocks of avenin components. These blocks can be used to assess the intrapopulation genetic diversity of oat varieties, as well as to search for adaptively significant and economically valuable traits linked to them.

The purpose is to study the varietal characteristics of the photosynthetic efficiency of oats when applying fertilizers in Western Siberia. Methods. The research was carried out on an experimental field located in the northern forest-steppe of the Trans-Urals. Photosynthetic potential and net productivity of photosynthesis were determined by the Nichiporovich’s method. Results. The nature of the formation of the assimilation surface and the accumulation of biomass of oat varieties Otrada, Talisman and Foma at different levels of mineral nutrition was studied. The photosynthetic potential and the net productivity of photosynthesis are calculated. The share of the influence of weather conditions, agrophone and variety on the studied indicators is determined. The Foma variety was characterized by maximum leafiness, compared to the Talisman and Otrada. On a natural agrophone, its assimilation surface in the sweeping phase reached 36.0 ± 5.5 thousand m2, at Otrada and Talisman – 28.1 and 24.0 thousand m2 per 1 ha of sowing. A very strong variability of the leaf surface area in Otrada was revealed (Cv = 24 %). The use of mineral fertilizers enhances the development of the leaf apparatus by 44–50 % and causes competition between oat plants. A close positive correlation was revealed between the leaf area in the sweeping phase and yield (r = 0.72). The area of the assimilation surface of oats is affected by: weather conditions – 37 %; nutrition level – 30 % and variety – 25 %. It was found that the Otrada and Foma varieties have the same accumulation dynamics and form the maximum photosynthetic biomass by the sweeping phase. The nature of biomass accumulation depends on weather conditions by 39 %; on mineral fertilizers by 29 % and on the genotype of the variety by 17 %. The productivity of the studied varieties is formed by increasing the assimilation surface, rather than increasing the efficiency of photosynthesis. Scientific novelty. For the first time, the photosynthetic potential was determined and the efficiency of the net photosynthesis productivity of oat varieties of the Tyumen selection was revealed. The share of the influence of various factors in the formation of the oat crop in the conditions of the Northern Trans-Urals has been established.

The article deals with the issues of cartographic support development for effective decision making support in the field of waste and secondary resources management. The analysis of foreign experience is adduced, the database of the sources of recyclable materials is collected and illustrated by an example of the Arctic zone of the Russian Federation, some mapping results are presented. The presented technological devices can be used for the development of systems for data collecting, storing and processing in the field of environmental management.

Molecular and biochemical markers are used to analyze the intraspecific genetic diversity of crops. Prolamin-coding loci are highly effective for assessing this indicator. On the basis of the Laboratory of Varietal Seed Identification of the State Agrarian University of the Northern Trans-Urals, 18 varieties of common oat included in the State Register of Selection Achievements in the Tyumen Region from the 1930s to 2019 were studied by electrophoresis in 2018-2019. The aim of the work was to study the dynamics of the genetic diversity of oat varieties at avenin-coding loci. For the analysis, 100 grains of each variety were used. Electrophoresis was carried out in vertical plates of 13.2 % polyacrylamide gel at a constant voltage of 500 V for 4.0-4.5 h. It was found that 44.4 % of the varieties are heterogeneous, each consisting of two biotypes. For three loci, 20 alleles were identified, 10 of which were detected for the first time. The allele frequency of avenin-coding loci varied with time. In the process of variety exchange, alleles that are characteristic of varieties of non-Russian origin were replaced by alleles present in domestic varieties and then in the varieties developed by local breeding institutions. The following alleles had the highest frequency in Tyumen varieties: Avn A4 (50.0 %), A2 (25.0 %), Avn B4 (50.0 %), Bnew6 (37.5 %), Avn C1 (37.5 %), C2 and C5 (25.0 %). These alleles are of great value as markers of agronomically and adaptively important characters for the region in question. The amount of genetic diversity of oats varied with time from 0.33 in 1929-1950 to up to 0.75 in 2019. The high value of genetic diversity in modern breeding varieties of the Scientific Research Institute of Agriculture of the Northern Trans-Urals and an increase in this indicator over the past 20 years are associated with the use of genetically heterogeneous source material in the breeding process. This allowed obtaining varieties with high adaptive potentials in the natural climatic conditions of the region.Для анализа внутривидового генетического разнообразия сельскохозяйственных культур применяются разнообразные молекулярные и биохимические маркеры. Высокой эффективностью при оценке этого показателя обладают проламин-кодирующие локусы. На базе лаборатории сортовой идентификации семян Государственного аграрного университета Северного Зауралья в 2018–2019 гг. методом электрофореза исследованы 18 сортов овса посевного, включенных в Государственный реестр селекци- онных достижений по Тюменской области с 1930-х гг. до 2019 г. Целью работы было изучить динамику генетического разнообразия сортов по авенин-кодирующим локусам. Для анализа использовали по 100 зерновок каждого сорта. Электрофорез проводили в вертикальных пластинах 13.2 % полиакриламидного геля при постоянном напряжении 500 В в течение 4.0–4.5 ч. Установлено, что 44.4 % сортов гетерогенны и состоят из двух биотипов. Для трех локусов идентифицировано 20 аллелей, 10 из которых выявлены впервые. Частота встречаемости аллелей авенин-кодирующих локусов изменялась с течением времени. Аллели, характерные для сортов иностранного происхождения, в процессе сортосмены заместились аллелями, присутствующими в отечественных сортах, а затем в сортах местных селекционных учреждений. Наибольшую частоту встречаемости в сортах тюменской селекции имели аллели Avn A4 (50.0 %), A2 (25.0 %), Avn B4 (50.0 %), Bnew6 (37.5 %), Avn C1 (37.5 %), C2 и C5 (25.0 %). Эти аллели имеют большую ценность как маркеры хозяйственно ценных и адаптивно значимых признаков. Величина генетического разнообразия в сортах овса изменялась с течением времени от 0.33 в 1929–1950 гг. до 0.75 в 2019 г. Высокое значение генетического разнообразия в современных сортах селекции Научно-исследовательского института сельского хозяйства Северного Зауралья, а также увеличение этого показателя на протяжении последних 20 лет связаны с использованием в селекционном процессе генетически разнородного исход- ного материала. Это позволило получить сорта, обладающие высоким адаптивным потенциалом в при- родно-климатических условиях данного региона.

Alleles of avenin-coding loci were identified in 24 varieties of common oat origin from 6 different breeding centers of the Russian Federation. It was found that 33% of the studied varieties are homogeneous. Heterogeneous varieties contain from 2 to 10 biotypes. The analysis revealed 67 different genotypes, ten of which are found in the spectra of several varieties at once. Despite the presence of matching genotypes, all the studied varieties differ in the genetic formulas of avenin, the number and frequency of occurrence of biotypes. This makes it possible to effectively identify and distinguish oat varieties sown by Russian breeding using the method of prolamin electrophoresis. The analyzed groups of varieties are characterized by high values of genetic (0.54-0.79) and intra-population diversity, which indicates the stability of populations. The exception is varieties originating from the Irkutsk region, low values of genetic and intra-population diversity in the population of which may indicate the process of genetic erosion. Analysis of the frequency distribution of alleles of avenin-coding loci does not allow to determine whether oat varieties belong to certain breeding centers, which is associated with the introduction of the same genotypes into the breeding programs of different regions.

Organoids are self-organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here, we describe a method to establish long-term-expanding human airway organoids from broncho-alveolar resections or lavage material. The pseudostratified airway organoids consist of basal cells, functional multi-ciliated cells, mucus-producing secretory cells, and CC10-secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoids established from lung cancer resections and metastasis biopsies retain tumor histopathology as well as cancer gene mutations and are amenable to drug screening. Respiratory syncytial virus (RSV) infection recapitulates central disease features, dramatically increases organoid cell motility via the non-structural viral NS2 protein, and preferentially recruits neutrophils upon co-culturing. We conclude that human airway organoids represent versatile models for the in vitro study of hereditary, malignant, and infectious pulmonary disease.

Much of modern molecular and cell biology research relies on the ability to purify cell types by fluorescence activated cell sorting (FACS). FACS typically relies on the ability to label cell types of interest with antibodies or fluorescent transgenic constructs. However, antibody availability is often limited, and genetic manipulation is labor intensive or impossible in the case of primary human tissue. To date, no systematic method exists to enrich for cell types without a priori knowledge of cell type markers. Here we propose GateID, a computational method that combines single-cell transcriptomics with FACS index sorting to purify cell types of choice, using only native cellular properties such as cell size, granularity and mitochondrial content. We validate GateID by purifying various cell types from the zebrafish kidney marrow and the human pancreas to high purity without resorting to specific antibodies or transgenes.

Organoids are self-organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here we describe a method to establish long-term-expanding human airway organoids from broncho-alveolar biopsies or lavage material. The pseudostratified airway organoid epithelium consists of basal cells, functional multi-ciliated cells, mucus-producing goblet cells, and CC10-secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoid culture conditions also allow gene editing as well as the derivation of various types of lung cancer organoids. Respiratory syncytial virus (RSV) infection recapitulated central disease features and dramatically increases organoid cell motility, found to be driven by the non-structural viral NS2 protein. We conclude that human airway organoids represent versatile models for the in vitro study of hereditary, malignant, and infectious pulmonary disease.

A new direction of agribusiness has been formed in the modern agricultural industry. This is expressed in the appearance of highly specialized enterprises working in the field of production of high-quality seeds of agricultural crops. The use of electrophoresis method in varietal identification is a new technology in domestic agribusiness. The purpose of the research was to study the electrophoresis role of prolamins in varietal identification of crops for high-quality seed material. The laboratory of varietal identification of seeds analyzed 47 varieties of oats Russian selection. On the basis of data on the component composition of avenin, varieties with a high level of intersort genetic differences were identified: Megion, Fobos, Local (K-8427), Uspekh, Otrada, Pushkinskij. Groups of samples with identical component composition of prolamins were found. Their genetic formulas of avenin have the following form: Avn A2B4C2, Avn A4B4C2, Avn A2B4C1 or Avn A2B1C3. It is established that the method of electrophoresis of oat prolamins allows effectively distinguish varieties belonging to the same variety and indistinguishable by morphological features. Implementation of a system of regular laboratory control of purity and compliance of original and reproductive oats seeds by electrophoresis of prolamins is necessary to improve the competitiveness of Russian grain production in the world market.

We present a protocol for visualizing and quantifying single mRNA molecules in mammalian (mouse and human) tissues. In the approach described here, sets of about 50 short oligonucleotides, each labeled with a single fluorophore, are hybridized to target mRNAs in tissue sections. Each set binds to a single mRNA molecule and can be detected by fluorescence microscopy as a diffraction-limited spot. Tissue architecture is then assessed by counterstaining the sections with DNAdye (DAPI), and cell borders can be visualized with a dye-coupled antibody. Spots are detected automatically with custom-made software, which we make freely available. The mRNA molecules thus detected are assigned to single cells within a tissue semiautomatically by using a graphical user interface developed in our laboratory. In this protocol, we describe an example of quantitative analysis of mRNA levels and localization in mouse small intestine. The procedure (from tissue dissection to obtaining data sets) takes 3 d. Data analysis will require an additional 3–7 d, depending on the type of analysis.

The oat is one of the most widely cultivated crops in Western Siberia. One of the factors for obtaining stable grain crops is the use of high-quality seeds for sowing. To control the varietal purity and constancy of the biotypic composition of cereal varieties, the method of electrophoresis of storage proteins - prolamins is successfully used. The aim of the study was to assess the effectiveness of the prolamin electrophoresis method for laboratory varietal control of oat using the example of varieties cultivated in the Tyumen region. Eighteen varieties of oats were analyzed, which are included in the State register of selection achievements in the region from 1929 to 2019. It was found that of the 26 genotypes, only 11 were sort-specific. Seven groups of varieties with identical types of prolamin spectra were found. The number of samples with individual types of avenin spectra at different time periods was not the same. Until 1970, from 25.0 to 33.3% of genotypes were characterized by variety-specific spectra. Later, with the advent of new varieties in the region, the number of genotypes with individual types of spectra increased and reached 100.0% by 2019. This makes it possible to accurately distinguish modern varieties of oats from each other, to carry out varietal identification and assessment of varietal purity. It is necessary to have up-to-date databases of reference spectra of varieties cultivated in the region and, in the case of varieties with identical types of spectra, assess varietal affiliation and varietal purity using other marker systems.

We present a protocol for visualizing and quantifying single mRNA molecules in mammalian (mouse and human) tissues. In the approach described here, sets of about 50 short oligonucleotides, each labeled with a single fluorophore, are hybridized to target mRNAs in tissue sections. Each set binds to a single mRNA molecule and can be detected by fluorescence microscopy as a diffraction-limited spot. Tissue architecture is then assessed by counterstaining the sections with DNA dye (DAPI), and cell borders can be visualized with a dye-coupled antibody. Spots are detected automatically with custom-made software, which we make freely available. The mRNA molecules thus detected are assigned to single cells within a tissue semiautomatically by using a graphical user interface developed in our laboratory. In this protocol, we describe an example of quantitative analysis of mRNA levels and localization in mouse small intestine. The procedure (from tissue dissection to obtaining data sets) takes 3 d. Data analysis will require an additional 3-7 d, depending on the type of analysis.

SummaryProliferation of the self-renewing epithelium of the gastric corpus occurs almost exclusively in the isthmus of the glands, from where cells migrate bidirectionally toward pit and base. The isthmus is therefore generally viewed as the stem cell zone. We find that the stem cell marker Troy is expressed at the gland base by a small subpopulation of fully differentiated chief cells. By lineage tracing with a Troy-eGFP-ires-CreERT2 allele, single marked chief cells are shown to generate entirely labeled gastric units over periods of months. This phenomenon accelerates upon tissue damage. Troy+ chief cells can be cultured to generate long-lived gastric organoids. Troy marks a specific subset of chief cells that display plasticity in that they are capable of replenishing entire gastric units, essentially serving as quiescent “reserve” stem cells. These observations challenge the notion that stem cell hierarchies represent a “one-way street.”

Leucine-rich repeat-containing G-protein coupled receptor 5-positive (Lgr5(+)) stem cells reside at crypt bottoms of the small and large intestine. Small intestinal Paneth cells supply Wnt3, EGF, and Notch signals to neighboring Lgr5(+) stem cells. Whereas the colon lacks Paneth cells, deep crypt secretory (DCS) cells are intermingled with Lgr5(+) stem cells at crypt bottoms. Here, we report regenerating islet-derived family member 4 (Reg4) as a marker of DCS cells. To investigate a niche function, we eliminated DCS cells by using the diphtheria-toxin receptor gene knocked into the murine Reg4 locus. Ablation of DCS cells results in loss of stem cells from colonic crypts and disrupts gut homeostasis and colon organoid growth. In agreement, sorted Reg4(+) DCS cells promote organoid formation of single Lgr5(+) colon stem cells. DCS cells can be massively produced from Lgr5(+) colon stem cells in vitro by combined Notch inhibition and Wnt activation. We conclude that Reg4(+) DCS cells serve as Paneth cell equivalents in the colon crypt niche.

By employing the sensitivity of single molecule fluorescent in situ hybridisation (smFISH) we have precisely quantified the levels and defined the temporal and spatial distribution of Hedgehog signalling activity during embryonic skin development, and uncovered that there is a Hedgehog signalling gradient along the proximal-distal axis of developing hair follicles. In order to explore the contribution of Hedgehog receptors Ptch1 and Ptch2 in establishing the epidermal signalling gradient, we quantitated the level of pathway activity generated in Ptch1 and Ptch1;Ptch2-deficient skin and defined the contribution of each receptor to regulation of the levels of Hedgehog signalling identified in wild-type skin. Moreover, we show that both the cellular phenotype and level of pathway activity featured in Ptch1;Ptch2-deficient cells faithfully recapitulates the Peak level of endogenous Hedgehog signalling detected at the base of developing follicles, where the concentration of endogenous Shh is predicted to be highest. Taken together, these data demonstrate that both Ptch1 and Ptch2 play a crucial role in sensing the concentration of Hedgehog ligand and regulating the appropriate dose-dependent response.

Summary Adult mitotic tissues like the intestine, skin, and blood undergo constant turnover throughout the life of an organism. Knowing the identity of the stem cell is crucial to understanding tissue homeostasis and its aberrations upon disease. Here we present a computational method for the derivation of a lineage tree from single-cell transcriptome data. By exploiting the tree topology and the transcriptome composition, we establish StemID, an algorithm for identifying stem cells among all detectable cell types within a population. We demonstrate that StemID recovers two known adult stem cell populations, Lgr5+ cells in the small intestine and hematopoietic stem cells in the bone marrow. We apply StemID to predict candidate multipotent cell populations in the human pancreas, a tissue with largely uncharacterized turnover dynamics. We hope that StemID will accelerate the search for novel stem cells by providing concrete markers for biological follow-up and validation., Graphical Abstract, Highlights • StemID infers the lineage tree and identifies stem cells from single-cell mRNA-seq data • Direct links of stem cells to distinct sub-types reflect transcriptome plasticity • The permissive stem cell transcriptome is characterized by high entropy • StemID infers candidate multipotent cell populations in the human pancreas, Grün et al. developed an algorithm, StemID, for the derivation of cell lineage trees and identification of stem cells from single-cell mRNA sequencing data. StemID successfully recovered known adult stem cell populations from the small intestine and bone marrow and was then used to predict a novel multipotent cell population in the human pancreas.

Lgr5 stem cells reside at small intestinal crypt bottoms, generating both the enterocyte and secretory lineage. Entry into the latter epithelial lineage requires silencing of Notch signaling. The Notch ligand Dll1 is strongly up-regulated in a small subset of immediate stem cell daughters. Lineage tracing utilizing a novel Dll1GFP-ires-CreERT2 knock-in mouse reveals that single Dll1high cells generate small, short-lived clones of all four secretory cell types. In culture, sorted Dll1high cells can form long-lived organoids when briefly exposed to Wnt3A. When Dll1 cells are genetically marked prior to tissue damage, significant numbers of stem cell tracing events occur. Lineage specification therefore occurs already in the earliest stem cell daughters through Notch lateral inhibition. Yet, specified secretory progenitors display plasticity and can regain stemness upon tissue damage.

A cell's function is influenced by the environment, or niche, in which it resides. Studies of niches usually require assumptions about the cell types present, which impedes the discovery of new cell types or interactions. Here we describe ProximID, an approach for building a cellular network based on physical cell interaction and single-cell mRNA sequencing, and show that it can be used to discover new preferential cellular interactions without prior knowledge of component cell types. ProximID found specific interactions between megakaryocytes and mature neutrophils and between plasma cells and myeloblasts and/or promyelocytes (precursors of neutrophils) in mouse bone marrow, and it identified a Tac1+ enteroendocrine cell-Lgr5+ stem cell interaction in small intestine crypts. This strategy can be used to discover new niches or preferential interactions in a variety of organs.

Introduction Cancer formation is initiated by a single cell that acquires sequential mutations, eventually resulting in a metastasizing carcinoma. Throughout this process numerous clones arise, each with their own unique genetic composition. All these clones have slightly different fitnesses and compete with each other; this process of competition (clonal dynamics) plays an important role in cancer progression and determines the genetic composition of the tumour as a whole. Material and methods To understand these clonal dynamics we have developed a viral lineage tracing system, which allows us to simultaneously track the clonal dynamics of thousands of unique clones. We applied this system to an organoid model of colon carcinoma progression, which consists of WT, APC -/- (A), APC -/- P53 -/- (AP), APC -/- P53 -/- KRAS G12D (APK) and APC -/- P53 -/- KRAS G12D SMAD4 -/- (APKS) organoid lines, which mimics colon carcinoma progression in patients. Results and discussions We found that clonal dynamics change dramatically throughout progression from A/AP to APK/APKS, where there is fast and stringent selection in A/AP organoid lines and slower selection in APK/APKS organoids lines. Furthermore Whole Genome Sequencing (WGS) of the organoids revealed that Copy Number Variation (CNV) patterns are deterministic in the A/AP organoids, with a strong preference for losses of chromosome 4 and chromosome 18, and much more varied in APK/APKS organoids. Finally, through single cell whole genome sequencing it was possible to identify the sequence of CNV events through which unique genetic clones arise in the APKS mutant organoids. Conclusion In this study, we find reproducible differences in clonal dynamics between adenoma and carcinoma like organoids. These differences in clonal dynamics are in line with current views on colon carcinoma progression, where early in the progression of the tumour several large-scale clonal expansions occur, while late in progression clonal dynamics are less extreme.

The Rho family GTPases Cdc42 and Rac1 are critical regulators of the actin cytoskeleton and are essential for skin and hair function. Wiskott-Aldrich syndrome family proteins act downstream of these GTPases, controlling actin assembly and cytoskeletal reorganization, but their role in epithelial cells has not been characterized in vivo. Here, we used a conditional knockout approach to assess the role of neural Wiskott-Aldrich syndrome protein (N-WASP), the ubiquitously expressed Wiskott-Aldrich syndrome-like (WASL) protein, in mouse skin. We found that N-WASP deficiency in mouse skin led to severe alopecia, epidermal hyperproliferation, and ulceration, without obvious effects on epidermal differentiation and wound healing. Further analysis revealed that the observed alopecia was likely the result of a progressive and ultimately nearly complete block in hair follicle (HF) cycling by 5 months of age. N-WASP deficiency also led to abnormal proliferation of skin progenitor cells, resulting in their depletion over time. Furthermore, N-WASP deficiency in vitro and in vivo correlated with decreased GSK-3beta phosphorylation, decreased nuclear localization of beta-catenin in follicular keratinocytes, and decreased Wnt-dependent transcription. Our results indicate a critical role for N-WASP in skin function and HF cycling and identify a link between N-WASP and Wnt signaling. We therefore propose that N-WASP acts as a positive regulator of beta-catenin-dependent transcription, modulating differentiation of HF progenitor cells.

Adult neurogenesis in the murine dentate gyrus occurs in a specialized microenvironment that sustains the generation of neurons during life. To fully understand adult neurogenesis, it is essential to determine the neural stem cell (NSC) and progenitor developmental stages, their molecular determinants, and the niche cellular and molecular composition. We report on a single-cell RNA sequencing study of the hippocampal niche, performed by isolating all the non-neuronal cell populations. Our analysis provides a comprehensive description of the dentate gyrus cells, and it allows the identification of exclusive cell-type-specific markers. We define the developmental stages and transcriptional dynamics of NSCs and progenitors, and we find that, while NSCs represent a heterogeneous cellular continuum, progenitors can be grouped into distinct subtypes. We determine the oligodendrocyte lineage and transcriptional dynamics, and we describe the microglia transcriptional profile and activation state. The combined data constitute a valuable resource to understand regulatory mechanisms of adult neurogenesis. Artegiani et al. perform single-cell sequencing analysis of the dentate gyrus, and they identify molecular features and lineage relations of the cell types present in the niche.

Understanding the development and function of an organ requires the characterization of all of its cell types. Traditional methods for visualizing and isolating subpopulations of cells are based on messenger RNA or protein expression of only a few known marker genes. The unequivocal identification of a specific marker gene, however, poses a major challenge, particularly if this cell type is rare. Identifying rare cell types, such as stem cells, short-lived progenitors, cancer stem cells, or circulating tumour cells, is crucial to acquire a better understanding of normal or diseased tissue biology. To address this challenge we first sequenced the transcriptome of hundreds of randomly selected cells from mouse intestinal organoids, cultured self-organizing epithelial structures that contain all cell lineages of the mammalian intestine. Organoid buds, like intestinal crypts, harbour stem cells that continuously differentiate into a variety of cell types, occurring at widely different abundances. Since available computational methods can only resolve more abundant cell types, we developed RaceID, an algorithm for rare cell type identification in complex populations of single cells. We demonstrate that this algorithm can resolve cell types represented by only a single cell in a population of randomly sampled organoid cells. We use this algorithm to identify Reg4 as a novel marker for enteroendocrine cells, a rare population of hormone-producing intestinal cells. Next, we use Reg4 expression to enrich for these rare cells and investigate the heterogeneity within this population. RaceID confirmed the existence of known enteroendocrine lineages, and moreover discovered novel subtypes, which we subsequently validated in vivo. Having validated RaceID we then applied the algorithm to ex vivo-isolated Lgr5-positive stem cells and their direct progeny. We find that Lgr5-positive cells represent a homogenous abundant population of stem cells mixed with a rare population of Lgr5-positive secretory cells. We envision broad applicability of our method for discovering rare cell types and the corresponding marker genes in healthy and diseased organs.

Regulation of the assembly and expression of actin is of major importance in diverse cellular functions such as motility and adhesion and in defining cellular and tissue architecture. These biological processes are controlled by changing the balance between polymerized (F) and soluble (G) actin. Previous studies have indicated the existence of an autoregulatory pathway that links the state of assembly and expression of actin, resulting in the reduction of actin synthesis after actin filaments are depolymerized. We have employed the marine toxins swinholide A and latrunculin A, both disrupting the organization of the actin-cytoskeleton, to determine whether this autoregulatory response is activated by a decrease in the level of polymerized actin or by an increase in monomeric actin concentrations in the cell. We showed that in cells treated with swinholide A the level of filamentous actin is decreased, and using a reversible cross-linking reagent, we found that actin dimers are formed. Latrunculin A also disassembled actin filaments, but produced monomeric actin, followed by a reduction in actin and vinculin expression, while swinholide A treatment elevated the synthesis of these proteins. In cells treated with both latrunculin A and swinholide A, dimeric actin was formed, and actin and vinculin synthesis were higher than in control cells. These results suggest that the substrate that confers an autoregulated reduction in actin expression is monomeric actin, and when its level is decreased by dimeric actin formation, actin synthesis is increased. J. Cell. Biochem. 65:469–478. © 1997 Wiley-Liss Inc.

To investigate the transition from non-cancerous to metastatic from a physical sciences perspective, the Physical Sciences–Oncology Centers (PS-OC) Network performed molecular and biophysical comparative studies of the non-tumorigenic MCF-10A and metastatic MDA-MB-231 breast epithelial cell lines, commonly used as models of cancer metastasis. Experiments were performed in 20 laboratories from 12 PS-OCs. Each laboratory was supplied with identical aliquots and common reagents and culture protocols. Analyses of these measurements revealed dramatic differences in their mechanics, migration, adhesion, oxygen response, and proteomic profiles. Model-based multi-omics approaches identified key differences between these cells' regulatory networks involved in morphology and survival. These results provide a multifaceted description of cellular parameters of two widely used cell lines and demonstrate the value of the PS-OC Network approach for integration of diverse experimental observations to elucidate the phenotypes associated with cancer metastasis., National Cancer Institute (U.S.) (Grant U54CA143874)

The human pathogens enteropathogenic Escherichia coli (EPEC) and vaccinia virus trigger actin assembly in host cells by activating the host adaptor Nck and the actin nucleation promoter neural Wiskott-Aldrich syndrome protein (N-WASP). EPEC translocates effector molecules into host cells via type III secretion, and the interaction between the translocated intimin receptor (Tir) and the bacterial membrane protein intimin stimulates Nck and N-WASP recruitment, leading to the formation of actin pedestals beneath adherent bacteria. Vaccinia virus also recruits Nck and N-WASP to generate actin tails that promote cell-to-cell spread of the virus. In addition to Nck and N-WASP, WASP-interacting protein (WIP) localizes to vaccinia virus tails, and inhibition of actin tail formation upon ectopic expression of WIP mutants led to the suggestion that WIP is required for this process. Similar studies of WIP mutants, however, did not affect the ability of EPEC to form actin pedestals, arguing against an essential role for WIP in EPEC-induced actin assembly. In this study, we demonstrate that Nck and N-WASP are normally recruited by vaccinia virus and EPEC in the absence of WIP, and neither WIP nor the WIP family members CR16 and WIRE/WICH are essential for pathogen induced actin assembly. In addition, although Nck binds EPEC Tir directly, N-WASP is required for its localization during pedestal formation. Overall, these data highlight similar pathogenic strategies shared by EPEC and vaccinia virus by demonstrating a requirement for both Nck and N-WASP, but not WIP or WIP family members in pathogen-induced actin assembly.

Determining the molecular identities of adult stem cells requires novel technologies for sensitive transcript detection in tissues. In mouse intestinal crypts, lineage-tracing studies suggested that different genes uniquely mark spatially distinct stem-cell populations, residing either at crypt bases or at position +4, but a detailed analysis of their spatial co-expression has not been feasible. Here we apply three-color single molecule fluorescence in-situ hybridization to study a comprehensive panel of intestinal stem-cell markers during homeostasis, aging and regeneration. We find that the expression of all markers overlap at crypt-base-cells. This co-expression includes Lgr5, Bmi1 and mTert, genes previously suggested to mark distinct stem cells. Strikingly, Dcamkl-1 tuft cells, distributed throughout the crypt axis, co-express Lgr5 and other stem cell markers that are otherwise confined to crypt bases. We also detect significant changes in the expression of some of the markers following irradiation, suggesting their potential role in the regeneration process. Our approach can enable the sensitive detection of putative stem cells in other tissues and in tumours, guiding complementary functional studies to evaluate their stem-cell properties.

The function of the NF2 tumor suppressor merlin has remained elusive despite increasing evidence for its role in actin cytoskeleton reorganization. The closely related ERM proteins (ezrin, radixin, and moesin) act as linkers between the cell membrane and cytoskeleton, and have also been implicated as active actin reorganizers. We report here that merlin and the ERMs can interact with and regulate N-WASP, a critical regulator of actin dynamics. Merlin and moesin were found to inhibit N-WASP-mediated actin assembly in vitro, a function that appears independent of their ability to bind actin. Furthermore, exogenous expression of a constitutively active ERM inhibits N-WASP-dependent Shigella tail formation, suggesting that the ERMs may function as inhibitors of N-WASP function in vivo. This novel function of merlin and the ERMs illustrates a mechanism by which these proteins directly exert their effects on actin reorganization and also provides new insight into N-WASP regulation.

Monomeric (G) actin was shown to be involved in inhibiting its own synthesis by an autoregulatory mechanism that includes enhanced degradation of the actin mRNA [Bershadsky et al., 1995; Lyubimova et al., 1997]. We show that the 3'-untranslated region (3'-UTR) of beta-actin mRNA, but not its 5'-untranslated region, is important for this regulation. The level of full-length beta-actin mRNA in cells was reduced when actin filaments were depolymerized by treatment with latrunculin A and elevated when actin polymerization was induced by jasplakinolide. By contrast, the level of actin mRNA lacking the 3'-UTR remained unchanged when these drugs modulated the dynamics of actin assembly in the cell. Moreover, the transfection of cells with a construct encoding the autoregulation-deficient form of beta-actin mRNA led to very high levels of actin expression compared with transfection with the control actin construct and was accompanied by characteristic changes in cell morphology and the structure of the actin cytoskeleton. These results suggest that the autoregulatory mechanism working via the 3'-UTR of actin mRNA is involved in controlling the maintenance of a defined pool of actin monomers that could be necessary for the proper organization of the microfilament system and the cytoskeleton-mediated signaling.

Background The adhesion of cells to the extracellular matrix (ECM) generates transmembrane signals that affect cell proliferation, differentiation and survival. These signals are triggered by interactions between integrin and the ECM and involve tyrosine phosphorylation of specific proteins, including focal adhesion kinase (FAK) and paxillin, and the assembly of focal adhesions and actin bundles. In matrix-adherent, serum-starved Swiss 3T3 cells, the system of focal adhesions and actin bundles is poorly developed, and the level of tyrosine phosphorylation of FAK and paxillin is low. A number of growth factors rapidly stimulate tyrosine phosphorylation of these proteins and the assembly of focal adhesions and actin bundles. Growth factors and adhesion to the ECM are both necessary for the subsequent transition of cells to the S-phase of the cell cycle. Results In serum-starved Swiss 3T3 cells, the disruption of microtubules by nocodazole or vinblastine, without the addition of external growth factors, induces the rapid assembly of focal adhesions and microfilament bundles, tyrosine phosphorylation of FAK and paxillin, and subsequent enhancement of DNA synthesis. All these effects require cell adhesion to the ECM and do not occur when cells are plated on substrates coated with poly-L-lysine or concanavalin A. Inhibitors of tyrosine phosphorylation and cell contractility also eliminate the effects of microtubule disruption on adhesion-dependent signal transduction. Conclusions In ECM-attached cells, microtubule disruption activates the integrin-dependent signaling cascade, which leads to the assembly of matrix adhesions and the induction of DNA synthesis. The increase in cell contractility is an indispensable intermediate step in this signaling process.

Summary Much of current molecular and cell biology research relies on the ability to purify cell types by fluorescence-activated cell sorting (FACS). FACS typically relies on the ability to label cell types of interest with antibodies or fluorescent transgenic constructs. However, antibody availability is often limited, and genetic manipulation is labor intensive or impossible in the case of primary human tissue. To date, no systematic method exists to enrich for cell types without a priori knowledge of cell-type markers. Here, we propose GateID, a computational method that combines single-cell transcriptomics with FACS index sorting to purify cell types of choice using only native cellular properties such as cell size, granularity, and mitochondrial content. We validate GateID by purifying various cell types from zebrafish kidney marrow and the human pancreas to high purity without resorting to specific antibodies or transgenes., Graphical Abstract, Highlights • GateID predicts non-intuitive FACS gates to purify cell types based on scRNA-seq data • Predicted gates can be normalized and used in an unlimited amount of experiments • Zebrafish hematopoietic and human pancreatic cell types can be enriched up to 100% • Live GateID-purified populations can be used for downstream analyses, By integrating single-cell transcriptomics data with FACS index sorting data, GateID can be used to set nonintuitive sorting gates to efficiently isolate pure, live populations of desired cell types from heterogenous mixtures without the need for identifying labels, such as antibodies or transgenic reporters.