Cell type discovery and representation in the era of high-content single cell phenotyping

Background

A fundamental characteristic of multicellular organisms is the specialization of functional cell types through the process of differentiation. These specialized cell types not only characterize the normal functioning of different organs and tissues, they can also be used as cellular biomarkers of a variety of different disease states and therapeutic/vaccine responses. In order to serve as a reference for cell type representation, the Cell Ontology has been developed to provide a standard nomenclature of defined cell types for comparative analysis and biomarker discovery. Historically, these cell types have been defined based on unique cellular shapes and structures, anatomic locations, and marker protein expression. However, we are now experiencing a revolution in cellular characterization resulting from the application of new high-throughput, high-content cytometry and sequencing technologies. The resulting explosion in the number of distinct cell types being identified is challenging the current paradigm for cell type definition in the Cell Ontology.

Results

In this paper, we provide examples of state-of-the-art cellular biomarker characterization using high-content cytometry and single cell RNA sequencing, and present strategies for standardized cell type representations based on the data outputs from these cutting-edge technologies, including “context annotations” in the form of standardized experiment metadata about the specimen source analyzed and marker genes that serve as the most useful features in machine learning-based cell type classification models. We also propose a statistical strategy for comparing new experiment data to these standardized cell type representations.

Conclusion

The advent of high-throughput/high-content single cell technologies is leading to an explosion in the number of distinct cell types being identified. It will be critical for the bioinformatics community to develop and adopt data standard conventions that will be compatible with these new technologies and support the data representation needs of the research community. The proposals enumerated here will serve as a useful starting point to address these challenges.

     

Fine root and litterfall dynamics of three Korean pine (Pinus koraiensis) forests along an altitudinal gradient

Background and aims

Fine root and aboveground litterfall, two large fluxes of nutrients and carbon in the forest ecosystems, are key processes to be considered in efforts of measuring, modeling and predicting soil carbon sequestration.

Methods

We used sequential coring and litter trap to measure seasonal dynamics of fine root and litterfall in three Korean pine dominated forests along an altitudinal gradient in the Changbai Mountain during the 2012 growing season.

Results

Fine root biomass decreased significantly while necromass increased remarkably with altitude. Patterns and amounts of fine root production and mortality varied among forest types. Litterfall decreased significantly with altitude, whereas forest floor mass increased. Carbon inputs through fine root mortality and litterfall decreased significantly with altitude while carbon storage of fine root mass did not differ among forest types and carbon storage of forest floor mass was significantly larger in higher altitudinal forests due to lower turnover rates.

Conclusions

This study provided an insight into the variations of fine root and litterfall dynamics among three Korean pine forests which were associated with different vegetation traits and environmental conditions, and also the quantification of carbon fluxes through fine root mortality and litterfall for estimating carbon budget of temperate forest.     

Cadmium uptake,localization and stress-induced morphogenic response in the fern Pteris vittata

Cadmium uptake, tissue localization and structural changes induced at cellular level are essential to understand Cd tolerance in plants. In this study we have exposed plants of Pteris vittata to different concentrations of CdCl₂ (0, 30, 60, 100 μM) to evaluate the tolerance of the fern to cadmium. Cadmium content determination and its histochemical localization showed that P. vittata not only takes up, but also transports and accumulates cadmium in the aboveground tissues, delocalizing it mainly in the less bioactive tissues of the frond, the trichomes and the scales. Cadmium tolerance in P. vittata was strictly related to morphogenic response induced by the metal itself in the root system. Adaptive response regarded changes of the root apex size, the developmental pattern of root hairs, the differentiation of xylem elements and endodermal suberin lamellae. All the considered parameters suggest that, in our experimental conditions, 60 μM of Cd may represent the highest concentration that P. vittata can tolerate; indeed this Cd level even improves the absorbance features of the root and allows good transport and accumulation of the metal in the fronds. The results of this study can provide useful information for phytoremediation strategies of soils contaminated by Cd, exploiting the established ability of P. vittata to transport, delocalize in the aboveground biomass and accumulate polluting metals.     

An Integrated Approach for Landmark-Based Resistant Shape Analysis in 3D

The study of shape changes in morphology has seen a significant renovation in the last 20 years, particularly as a consequence of the development of geometric morphometric methods based on Cartesian coordinates of points. In order to extract information about shape differences when Cartesian coordinates are used, it is necessary to establish a common reference frame or system for all specimens to be compared. Therefore, a central issue in coordinate-based methods is which criterion should be used to align these configurations of points, since shape differences highly depend on those alignments. This is usually accomplished by aligning the configurations in a way that the sum of squared distances between coordinates of homologous points (landmarks) is minimized: the least-squares superimposition method. However, it is widely recognized that this method has some limitations when shape differences are not homogeneous across landmarks. Here we present an integrated approach for the resistant shape comparison of 3D landmark sets. It includes a new ordinary resistant Procrustes superimposition and its corresponding generalized resistant Procrustes version. In addition, they are combined with existing resistant multivariate statistical techniques for depicting the results. We demonstrate, by using both simulated and real datasets, that resistant Procrustes better detects and measures localized shape variation whenever present in up to half but one of the landmarks. The resistant Procrustes results are highly concordant with a priori biological information, and might dramatically improve the quality of inferences on patterns of shape variation.     

Diet of Eurasian lynx <Emphasis Type="Italic">Lynx lynx</Emphasis> in the northern Dinaric Mountains (Slovenia and Croatia)

The Eurasian lynx Lynx lynx (Linnaeus 1758) is an opportunistic predator that usually selects the smallest ungulate available. Its diet varies considerably among different regions; therefore it is important to study lynx diet in different parts of the species’ range. We studied the diet of lynx from the endangered Dinaric population in Slovenia and Croatia by analyzing lynx scats, prey remains, and stomach contents. Dinaric lynx mainly killed European roe deer Capreolus capreolus (0.64 frequency of occurrence, 79% of all consumed biomass), which were used more frequently during winter and spring. Ungulates were killed more often by adult males than by lynx of other age and sex groups. In contrast to studies from other regions, lynx in the northern Dinaric Mountains also frequently fed on the edible dormice Glis glis (0.18 frequency of occurrence, 7% of all consumed biomass). This large rodent appears to be an important alternative prey, especially for females and young lynx, and was the reason for the highest use of rodents reported so far for the Eurasian lynx. Edible dormice in Dinaric forests have highly variable numbers of active animals. Seasonal and possibly annual variation in dormouse availability obviously affects lynx diet. This is a rare example where variability in the availability of the alternative prey and not the preferred prey leads to the dietary shift. This study confirms the opportunistic nature of Eurasian lynx and the regional variability of its diet.     

Benefits of <Emphasis Type="Italic">Helicobacter pylori</Emphasis><Emphasis Type="Italic">cagE</Emphasis> genotyping in addition to <Emphasis Type="Italic">cagA</Emphasis> genotyping: a Bulgarian study

Associations of Helicobacter pylori cagE status with complex patient characteristics remain to be elucidated in Eastern Europe. The aim of this study was to assess the frequencies of cagE gene and cagA/cagE combinations in H. pylori strains from symptomatic Bulgarian patients and to improve cagA detection. cagA and cagE genotypes were evaluated in 219 patients with single-strain infections. In total, 84.9% of strains were cagA ⁺, while 68.5% were cagE ⁺. cagA ⁺, cagE ⁺, and cagA ⁺/cagE ⁺ strains were more prevalent in peptic ulcer (93.8%, 84.4%, and 84.4%) compared with nonulcer patients (81.3%, 61.9%, and 61.3%, respectively). In elderly patients, cagE ⁺ and cagA ⁺/cagE ⁺ strains were 1.9-fold more common than in the 12 children evaluated. Only 10% of the elderly subjects harbored low-virulence cagA ⁺/cagE ⁻ strains compared with 16.8% of adults and 41.7% of children. Intriguingly, prevalence of the cagA ⁺/cagE ⁻ genotype was 2.1-fold lower in men than in women, suggesting a higher frequency of more virulent strains in men. The presence of both cag genes and combinations was not linked to strain susceptibility to clarithromycin or metronidazole, place of residence, or prior therapy. Use of an extra primer pair increased cagA detection in 14.7% of 31 cagA ⁻ strains. In conclusion, use of a second primer pair for the cagA gene can be recommended in countries with common cagA ⁺ strains. Although both cag genes were linked to severe diseases in Bulgarian patients, the best discrimination of virulent strains was obtained by the cagA/cagE combination or by the cagE gene alone. cagE prevalence increased gradually with patient age, while the cagA ⁺/cagE ⁻ genotype, implying a disrupted cag pathogenicity island, was associated with both younger age and female gender.     

Oleic acid induces intracellular calcium mobilization, MAPK phosphorylation, superoxide production and granule release in bovine neutrophils

Oleic acid (OA) is a nonesterified fatty acid that is released into the blood during lipomobilization at the time of calving in cows, a period where increased risk of infection and acute inflammation is observed. These data suggest potential OA-mediated regulation of innate immune responses. In the present study, we assessed the effects of OA on intracellular calcium release, ERK1/2 phosphorylation, superoxide production, CD11b expression and matrix metalloproteinase-9 (MMP-9) release in bovine neutrophils. Furthermore, the presence of GPR40, an OA receptor, was assessed by RT-PCR, immunoblotting and confocal microscopy. OA induced, in a dose-dependent manner, intracellular calcium mobilization, superoxide production and CD11b expression in bovine neutrophils; these effects were reduced by the intracellular chelating agent BAPTA-AM. OA also induced ERK2 phosphorylation and MMP-9 release. RT-PCR analysis detected mRNA expression of a bovine ortholog of the GPR40 receptor. Using a polyclonal antibody against human GPR40, we detected a protein of 31 kDa by immunoblotting that was localized predominately in the plasma membrane. The selective agonist of GPR40, GW9508, induced intracellular calcium mobilization and ERK2 phosphorylation. In conclusion, OA can modulate bovine neutrophil responses in an intracellular calcium-dependent manner; furthermore, these responses could be induced by GPR40 activation.     

The role of ABC proteins Aus1p and Pdr11p in the uptake of external sterols in yeast: dehydroergosterol fluorescence study

Uptake of external sterols in the yeast Saccharomyces cerevisiae is a multistep process limited to anaerobiosis or heme deficiency. It includes crossing the cell wall, insertion of sterol molecules into plasma membrane and their internalization and integration into intracellular membranes. We applied the fluorescent ergosterol analog dehydroergosterol (DHE) to monitor the initial steps of sterol uptake by three independent approaches: fluorescence spectroscopy, fluorescence microscopy and sterol quantification by HPLC. Using specific fluorescence characteristics of DHE we showed that the entry of sterol molecules into plasma membrane is not spontaneous but requires assistance of two ABC (ATP-binding cassette) pumps – Aus1p or Pdr11p. DHE taken up by uptake-competent hem1ΔAUS1PDR11 cells could be directly visualized by UV-sensitive wide field fluorescence microscopy. HPLC analysis of sterols revealed significant amounts of exogenous ergosterol and DHE (but not cholesterol) associated with uptake-deficient hem1Δaus1Δpdr11Δ cells. Fluorescent sterol associated with these cells did not show the characteristic emission spectrum of membrane-integrated DHE. The amount of cell-associated DHE was significantly reduced after enzymatic removal of the cell wall. Our results demonstrate that the yeast cell wall is actively involved in binding and uptake of ergosterol-like sterols.     

Structural-functional role of chloride in photosystem II

Chloride binding in photosystem II (PSII) is essential for photosynthetic water oxidation. However, the functional roles of chloride and possible binding sites, during oxygen evolution, remain controversial. This paper examines the functions of chloride based on its binding site revealed in the X-ray crystal structure of PSII at 1.9 ? resolution. We find that chloride depletion induces formation of a salt bridge between D2-K317 and D1-D61 that could suppress the transfer of protons to the lumen.