CAbiNet: joint clustering and visualization of cells and genes for single-cell transcriptomics.

A fundamental analysis task for single-cell transcriptomics data is clustering with subsequent visualization of cell clusters. The genes responsible for the clustering are only inferred in a subsequent step. Clustering cells and genes together would be the remit of biclustering algorithms, which are often bogged down by the size of single-cell data. Here we present 'Correspondence Analysis based Biclustering on Networks' (CAbiNet) for joint clustering and visualization of single-cell RNA-sequencing data. CAbiNet performs efficient co-clustering of cells and their respective marker genes and jointly visualizes the biclusters in a non-linear embedding for easy and interactive visual exploration of the data.

Vertical distribution of Candidatus Methylomirabilis and Methanoperedens in agricultural soils.

Candidatus Methylomirabilis-related bacteria conduct anaerobic oxidation of methane (AOM) coupling with NO2- reduction, and Candidatus Methanoperedens-related archaea perform AOM coupling with reduction of diverse electron acceptors, including NO3-, Fe (III), Mn (IV) and SO42-. Application of nitrogen fertilization favors the growth of these methanotrophs in agricultural fields. Here, we explored the vertical variations in community structure and abundance of the two groups of methanotrophs in a nitrogen-rich vegetable field via using illumina MiSeq sequencing and quantitative PCR. The retrieved Methylomirabilis-related sequences had 91.12%-97.32% identity to the genomes of known Methylomirabilis species, and Methanoperedens-related sequences showed 85.49%-97.48% identity to the genomes of known Methanoperedens species which are capable of conducting AOM coupling with reduction of NO3- or Fe (III). The Methanoperedens-related archaeal diversity was significantly higher than Methylomirabilis-related bacteria, with totally 74 and 16 operational taxonomic units, respectively. In contrast, no significant difference in abundance between the bacteria (9.19 × 103-3.83 × 105 copies g-1 dry soil) and the archaea (1.55 × 104-3.24 × 105 copies g-1 dry soil) was observed. Furthermore, the abundance of both groups of methanotrophs exhibited a strong vertical variation, which peaked at 30-40 and 20-30 cm layers, respectively. Soil water content and pH were the key factors influencing Methylomirabilis-related bacterial diversity and abundance, respectively. For the Methanoperedens-related archaea, both soil pH and ammonium content contributed significantly to the changes of these archaeal diversity and abundance. Overall, we provide the first insights into the vertical distribution and regulation of Methylomirabilis-related bacteria and Methanoperedens-related archaea in vegetable soils. KEY POINTS: • The archaeal diversity was significantly higher than bacterial. • There was no significant difference in the abundance between bacteria and archaea. • The abundance of bacteria and archaea peaked at 30-40 and 20-30 cm, respectively.

Upgraded CRISPR/Cas9 tools for tissue-specific mutagenesis in Drosophila.

CRISPR/Cas9 has emerged as a powerful technology for tissue-specific mutagenesis. However, tissue-specific CRISPR/Cas9 tools currently available in Drosophila remain deficient in three significant ways. First, many existing gRNAs are inefficient, such that further improvements of gRNA expression constructs are needed for more efficient and predictable mutagenesis in both somatic and germline tissues. Second, it has been difficult to label mutant cells in target tissues with current methods. Lastly, application of tissue-specific mutagenesis at present often relies on Gal4-driven Cas9, which hampers the flexibility and effectiveness of the system. Here, we tackle these deficiencies by building upon our previous CRISPR-mediated tissue-restricted mutagenesis (CRISPR-TRiM) tools. First, we significantly improved gRNA efficiency in somatic tissues by optimizing multiplexed gRNA design. Similarly, we also designed efficient dual-gRNA vectors for the germline. Second, we developed methods to positively and negatively label mutant cells in tissue-specific mutagenesis by incorporating co-CRISPR reporters into gRNA expression vectors. Lastly, we generated genetic reagents for convenient conversion of existing Gal4 drivers into tissue-specific Cas9 lines based on homology-assisted CRISPR knock-in. In this way, we expand the choices of Cas9 for CRISPR-TRiM analysis to broader tissues and developmental stages. Overall, our upgraded CRISPR/Cas9 tools make tissue-specific mutagenesis more versatile, reliable, and effective in Drosophila These improvements may be also applied to other model systems.

The Drosophila Trpm channel mediates calcium influx during egg activation.

Egg activation is the process in which mature oocytes are released from developmental arrest and gain competency for embryonic development. In Drosophila and other arthropods, eggs are activated by mechanical pressure in the female reproductive tract, whereas in most other species, eggs are activated by fertilization. Despite the difference in the trigger, Drosophila shares many conserved features with higher vertebrates in egg activation, including a rise of intracellular calcium in response to the trigger. In Drosophila, this calcium rise is initiated by entry of extracellular calcium due to opening of mechanosensitive ion channels and initiates a wave that passes across the egg prior to initiation of downstream activation events. Here, we combined inhibitor tests, germ-line-specific RNAi knockdown, and germ-line-specific CRISPR/Cas9 knockout to identify the Transient Receptor Potential (TRP) channel subfamily M (Trpm) as a critical channel that mediates the calcium influx and initiates the calcium wave during Drosophila egg activation. We observed a reduction in the proportion of eggs that hatched from trpm germ-line knockout mutant females, although eggs were able to complete some egg activation events including cell cycle resumption. Since a mouse ortholog of Trpm was recently reported also to be involved in calcium influx during egg activation and in further embryonic development, our results suggest that calcium uptake from the environment via TRPM channels is a deeply conserved aspect of egg activation.

Regulation of Trpm activation and calcium wave initiation during Drosophila egg activation.

The transition from a developmentally arrested mature oocyte to a developing embryo requires a series of highly conserved events, collectively known as egg activation. All of these events are preceded by a ubiquitous rise of intracellular calcium, which results from influx of external calcium and/or calcium release from internal storage. In Drosophila, this calcium rise initiates from the pole(s) of the oocyte by influx of external calcium in response to mechanical triggers. It is thought to trigger calcium responsive kinases and/or phosphatases, which in turn alter the oocyte phospho-proteome to initiate downstream events. Recent studies revealed that external calcium enters the activating Drosophila oocyte through Trpm channels, a feature conserved in mouse. The local entry of calcium raises the question of whether Trpm channels are found locally at the poles of the oocyte or are localized around the oocyte periphery, but activated only at the poles. Here, we show that Trpm is distributed all around the oocyte. This requires that it thus be specially regulated at the poles to allow calcium wave initiation. We show that neither egg shape nor local pressure is sufficient to explain this local activation of Trpm channels.

A calcium-mediated actin redistribution at egg activation in Drosophila.

Egg activation is the essential process in which mature oocytes gain the competency to proceed into embryonic development. Many events of egg activation are conserved, including an initial rise of intracellular calcium. In some species, such as echinoderms and mammals, changes in the actin cytoskeleton occur around the time of fertilization and egg activation. However, the interplay between calcium and actin during egg activation remains unclear. Here, we use imaging, genetics, pharmacological treatment, and physical manipulation to elucidate the relationship between calcium and actin in living Drosophila eggs. We show that, before egg activation, actin is smoothly distributed between ridges in the cortex of the dehydrated mature oocytes. At the onset of egg activation, we observe actin spreading out as the egg swells though the intake of fluid. We show that a relaxed actin cytoskeleton is required for the intracellular rise of calcium to initiate and propagate. Once the swelling is complete and the calcium wave is traversing the egg, it leads to a reorganization of actin in a wavelike manner. After the calcium wave, the actin cytoskeleton has an even distribution of foci at the cortex. Together, our data show that calcium resets the actin cytoskeleton at egg activation, a model that we propose to be likely conserved in other species.

Simultaneous methane mitigation and nitrogen removal by denitrifying anaerobic methane oxidation in lake sediments.

Methane (CH4) is a potent greenhouse gas, with lake ecosystems significantly contributing to its global emissions. Denitrifying anaerobic methane oxidation (DAMO) process, mediated by NC10 bacteria and ANME-2d archaea, links global carbon and nitrogen cycles. However, their potential roles in mitigating methane emissions and removing nitrogen from lake ecosystems remain unclear. This study explored the spatial variations in activities of nitrite- and nitrate-DAMO and their functional microbes in Changdanghu Lake sediments (Jiangsu Province, China). The results showed that although the average abundance of ANME-2d archaea (5.0 × 106 copies g-1) was significantly higher than that of NC10 bacteria (2.1 × 106 copies g-1), the average potential rates of nitrite-DAMO (4.59 nmol 13CO2 g-1 d-1) and nitrate-DAMO (5.01 nmol 13CO2 g-1 d-1) showed no significant difference across all sampling sites. It is estimated that nitrite- and nitrate-DAMO consumed approximately 6.46 and 7.05 mg CH4 m-2 d-1, respectively, which accordingly achieved 15.07-24.95 mg m-2 d-1 nitrogen removal from the studied lake sediments. Statistical analyses found that nitrite- and nitrate-DAMO activities were both significantly related to sediment nitrate contents and ANME-2d archaeal abundance. In addition, NC10 bacterial and ANME-2d archaeal community compositions showed significant correlations with sediment organic carbon content and water depth. Overall, this study underscores the dual roles of nitrite- and nitrate-DAMO processes in CH4 mitigation and nitrogen elimination and their key environmental impact factors (sediment organic carbon and inorganic nitrogen contents, and water depth) in shallow lake, enhancing the understanding of carbon and nitrogen cycles in freshwater aquatic ecosystems.

Effects of increased ozone on rice panicle morphology.

Ground-level ozone threatens rice production, which provides staple food for more than half of the world's population. Improving the adaptability of rice crops to ozone pollution is essential to ending global hunger. Rice panicles not only affect grain yield and grain quality but also the adaptability of plants to environmental changes, but the effects of ozone on rice panicles are not well understood. Through an open top chamber experiment, we investigated the effects of long-term and short-term ozone on the traits of rice panicles, finding that both long-term and short-term ozone significantly reduced the number of panicle branches and spikelets in rice, and especially the fertility of spikelets in hybrid cultivar. The reduction in spikelet quantity and fertility because of ozone exposure is caused by changes in secondary branches and attached spikelet. These results suggest the potential for effective adaptation to ozone by altering breeding targets and developing growth stage-specific agricultural techniques.

Spatio-temporal variations of activity of nitrate-driven anaerobic oxidation of methane and community structure of Candidatus Methanoperedens-like archaea in sediment of Wuxijiang river.

Nitrate-driven anaerobic oxidation of methane (AOM), catalyzing by Candidatus Methanoperedens-like archaea, is a new addition in the global CH4 cycle. This AOM process acts as a novel pathway for CH4 emission reduction in freshwater aquatic ecosystems; however, its quantitative importance and regulatory factors in riverine ecosystems are nearly unknown. Here, we examined the spatio-temporal changes of the communities of Methanoperedens-like archaea and nitrate-driven AOM activity in sediment of Wuxijiang River, a mountainous river in China. These archaeal community composition varied significantly among reaches (upper, middle, and lower reaches) and between seasons (winter and summer), but their mcrA gene diversity showed no significant spatial or temporal variations. The copy numbers of Methanoperedens-like archaeal mcrA genes were 1.32 × 105-2.47 × 107 copies g-1 (dry weight), and the activity of nitrate-driven AOM was 0.25-1.73 nmol CH4 g-1 (dry weight) d-1, which could potentially reduce 10.3% of CH4 emissions from rivers. Significant spatio-temporal variations of mcrA gene abundance and nitrate-driven AOM activity were found. Both the gene abundance and activity increased significantly from upper to lower reaches in both seasons, and were significantly higher in sediment collected in summer than in winter. In addition, the variations of Methanoperedens-like archaeal communities and nitrate-driven AOM activity were largely impacted by the sediment temperature, NH4+ and organic carbon contents. Taken together, both time and space scales need to be considered for better evaluating the quantitative importance of nitrate-driven AOM in reducing CH4 emissions from riverine ecosystems.

Food Security in China: A Brief View of Rice Production in Recent 20 Years.

Rice production affects the food security and socioeconomic status of over half the world's population. Rice-producing countries, however, are facing population growth, reduction in rice planting area, and global change. Understanding the trends of rice production and major determinants is key to regulating rice production. We thus analyzed the trends of rice production and related determinants in China from 2001 to 2021, revealing that the annual rice production (TRP) has risen steadily (r = 0.929, p < 0.0001) in recent 20 years. TRP in 2021 was 19.9% higher than that in 2001, which was primarily achieved by the increment of middle rice production (MRP). MRP increased by 46.2% from 2000 to 2018, and grain yield per unit area (GPA) was the largest in middle rice. The enhancement of GPAs is significantly correlated with the consumption of agricultural resources and the number of released rice cultivars, but variations exist. TRP and GPA vary in different provinces; Hunan (25 ± 2 megatons) and Xinjiang (8364 ± 806 kg/hectare) show the largest values, respectively. TRP could be further increased by 13.8% by improving MRP. The results suggest that rice production in China has a large potential to be further improved through regulations.

Ethylenediurea reduces grain chalkiness in hybrid rice cultivars under ambient levels of surface ozone in China.

High concentration of tropospheric ozone (O3) causes crop yield losses, which could be reduced by foliar application of ethylenediurea (EDU). Rice grain appearance is a major quality trait that determines the milling quality, white rice productivity and the market value. Grain chalkiness is one of the common defects that deteriorate the grain appearance in rice due to its negative effects on palatability and milling yield. Whether EDU could reduce grain chalkiness in rice which was usually increased by high concentration of O3 is not clarified. We report the grain chalkiness in 19 rice cultivars (CVs) of three types: indica (6 CVs), japonica (5 CVs) and hybrids (8 CVs), observed in an EDU application experiment in the field in China. The ambient O3 level as expressed by accumulated hourly O3 concentration over the threshold of 40 ppb (AOT40) for 80 days until maturity reached 12.8 ppm h at a near-by monitoring station. Fraction of the chalky grains (FCG) in the hybrid cultivars was 8% lower in EDU than that in the control treatments, whereas no significant effect of EDU on FCG was found in japonica or indica cultivars. The reduction of FCG due to EDU treatment in hybrid cultivars was attributed to the significant reduction of milky white grains followed by that of white belly grains. Thus, the application of EDU could ameliorate the decline of grain appearance quality in hybrid rice by decreasing the FCG and enhancing the fraction of perfect grains (FPG). Moreover, there were significant interactions between the EDU application and rice cultivars, indicating varietal difference in the protection of grain appearance quality by EDU. These results suggest the need for further studies on the mechanisms of the effects of EDU on grain chalkiness.

High-resolution 3D spatiotemporal transcriptomic maps of developing Drosophila embryos and larvae.

Drosophila has long been a successful model organism in multiple biomedical fields. Spatial gene expression patterns are critical for the understanding of complex pathways and interactions, whereas temporal gene expression changes are vital for studying highly dynamic physiological activities. Systematic studies in Drosophila are still impeded by the lack of spatiotemporal transcriptomic information. Here, utilizing spatial enhanced resolution omics-sequencing (Stereo-seq), we dissected the spatiotemporal transcriptomic changes of developing Drosophila with high resolution and sensitivity. We demonstrated that Stereo-seq data can be used for the 3D reconstruction of the spatial transcriptomes of Drosophila embryos and larvae. With these 3D models, we identified functional subregions in embryonic and larval midguts, uncovered spatial cell state dynamics of larval testis, and revealed known and potential regulons of transcription factors within their topographic background. Our data provide the Drosophila research community with useful resources of organism-wide spatiotemporally resolved transcriptomic information across developmental stages.

X-ray fluorescence microscopy scanning of Drosophila oocytes and eggs.

X-ray fluorescence microscopy (XFM) is a powerful tool for mapping and quantifying the spatial distribution of elemental composition of biological samples. Recently, it was reported that transition metal fluctuations occur during Drosophila reproduction, analogous to what is seen in mammals and nematodes, and may contribute to Drosophila female fertility. To further support XFM studies on Drosophila reproduction, we describe procedures for isolating oocytes and activated eggs and examining their elemental composition by XFM scanning and analysis. For complete details on the use and execution of this protocol, please refer to Hu et al. (2020).

Zinc Dynamics during Drosophila Oocyte Maturation and Egg Activation.

Temporal fluctuations in zinc concentration are essential signals, including during oogenesis and early embryogenesis. In mammals, zinc accumulation and release are required for oocyte maturation and egg activation, respectively. Here, we demonstrate that zinc flux occurs in Drosophila oocytes and activated eggs, and that zinc is required for female fertility. Our synchrotron-based X-ray fluorescence microscopy reveals zinc as the most abundant transition metal in Drosophila oocytes. Its levels increase during oocyte maturation, accompanied by the appearance of zinc-enriched intracellular granules in the oocyte, which depend on transporters. Subsequently, in egg activation, which mediates the transition from oocyte to embryo, oocyte zinc levels decrease significantly, as does the number of zinc-enriched granules. This pattern of zinc dynamics in Drosophila oocytes follows a similar trajectory to that in mammals, extending the parallels in female gamete processes between Drosophila and mammals and establishing Drosophila as a model for dissecting reproductive roles of zinc.

End-to-End syndrome differentiation of Yin deficiency and Yang deficiency in traditional Chinese medicine.

BACKGROUND AND OBJECTIVE: Yin and Yang, two concepts adapted from classical Chinese philosophy, play a diagnostic role in Traditional Chinese Medicine (TCM). The Yin and Yang in harmonious balance indicate health, whereas imbalances to either side indicate unhealthiness, which may result in diseases. Yin-yang disharmony is considered to be the cause of pathological changes. Syndrome differentiation of yin-yang is crucial to clinical diagnosis. It lays a foundation for subsequent medical judgments, including therapeutic methods, and formula, among many others. However, because of the complexities of the mechanisms and manifestations of disease, it is difficult to exactly point out which one, yin or yang, is disharmonious. There has been inadequate research conducted on syndrome differentiation of yin and yang from a computational perspective. In this study, we present a computational method, viz. an end-to-end syndrome differentiation of yin deficiency and yang deficiency. METHODS: Unlike most previous studies on syndrome differentiation, which use structured datasets, this study takes unstructured texts in medical records as its inputs. It models syndrome differentiation as a task of text classification. This study experiments on two state-of-the-art end-to-end algorithms for text classification, i.e. a classic convolutional neural network (CNN) and fastText. These two systems take the n-grams of several types of tokens as their inputs, including characters, terms, and words. RESULTS: When evaluated on a data set with 7326 modern medical records in TCM, it is observed that CNN and fastText generally give rise to comparable performances. The best accuracy rate of 92.55% comes from the system taking inputs as raw as n-grams of characters. It implies that one can build at least a moderate system for the differentiation of yin deficiency and yang deficiency even if he has no glossary or tokenizer at hand. CONCLUSIONS: This study has demonstrated the feasibility of using end-to-end text classification algorithms to differentiate yin deficiency and yang deficiency on unstructured medical records.