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Droplet-based single-cell RNA sequencing protocols have dramatically increased the throughput of single-cell transcriptomics studies. A key computational challenge when processing these data is to distinguish libraries for real cells from empty droplets. Here, we describe a new statistical method for calling cells from droplet-based data, based on detecting significant deviations from the expression profile of the ambient solution. Using simulations, we demonstrate that EmptyDrops has greater power than existing approaches while controlling the false discovery rate among detected cells. Our method also retains distinct cell types that would have been discarded by existing methods in several real data sets.

In recent years, several studies have been developed to understand the impact of fermentation on the final quality of coffee and have indicated that postharvest processing could be a determinant of quality. However, a trend has appeared as a scientific counterpoint, indicating that the interactions between soil, fruit, altitude, and slope exposures with respect to the Sun are important to understand the behavior of the microbiome in coffee. Studies on the microbiota of coffee have addressed its role during the fermentation process, however the knowledge of indigenous microorganisms harbored in fruits and soil of coffee trees growing in fields are essential, as they can contribute to fermentation. Therefore, the aim of this work was to evaluate the influence of topographic and edaphic factors on the bacterial and fungal communities present in the soil and in the fruits of Coffea arabica trees. Samples of fruits and soil were collected from different growing areas at different altitudes and soil conditions. The microbial DNA was extracted and sequenced. The results showed the contribution of environmental factors in the structure of bacterial and fungal communities. The richness, evenness and diversity of the mycobiome and bacteriome were higher in the soil than in the fruits, independent of altitude. In addition, coffee trees at higher altitudes tended to have more bacteria shared between the soil and fruits. The co-occurrence/co-exclusion network showed that bacteria-bacteria connections were greater in higher altitudes. On another hand, fungi-fungi and fungi-bacteria connections were higher in low altitudes. This was the first study that evaluates in deep the influence of environmental factors in the microbiota habiting fruits and soil coffee trees, which may affect the coffee beverage quality.

Agroecology aims to maintain ecosystem services by minimizing the impact of agriculture and promoting the use of biological potential. Arbuscular mycorrhizal fungi (AMF) are elements which are key to improving crop productivity and soil quality. It is pertinent to understand how agricultural management in the tropics affects the AMF spatio-temporal community composition, especially in crops of global importance, such as coffee (Coffea arabica L.). Soil and root samples were collected from three localities under three management systems (agroecological, conventional and forest fragment), during the phenological stages of coffee (flowering, grain filling, harvesting). Spores were extracted for morphological identification and molecular community analysis by PCR-DGGE. Dendrograms were prepared and the bands were sequenced and analyzed by bioinformatics. No differences were observed in the richness of morphospecies between management systems, localities and period, but little is known about tropical species. Molecular analysis showed that the agroecological management system was similar to natural forest and with a higher diversity indices than conventional management. Locality and period of sample affect AMF community composition. It is necessary to associate classical taxonomic evaluations with molecular biological techniques because different approaches can lead to different outcomes. This study contributes to the understanding of the impact of agriculture management systems on AMF and provides evidence that agroecology is a management system applicable to sustainable coffee production.

The genus Tulasnella often forms mycorrhizas with orchids and has worldwide distribution. Species of this genus are associated with a wide range of orchids, including endangered hosts. Initially, species identification relied mostly on morphological features and few cultures were preserved for later phylogenetic comparisons. In this study, a total of 50 Tulasnella isolates were collected from their natural sites in Minas Gerais, Brazil, cultured, and subjected to a phylogenetic analysis based on alignments of sequences of the internal transcribed spacer (ITS) of the nuclear ribosomal DNA. Our results, based on phylogeny, integrated with nucleotide divergence and morphology, revealed the diversity of isolated Tulasnella species, which included four new species, namely, Tulasnella brigadeiroensis , Tulasnella hadrolaeliae , Tulasnella orchidis and Tulasnella zygopetali . The conservation of these species is important due to their association with endangered orchid hosts and endemic features in the Brazilian Atlantic Forest.

Post-harvest processing and microbial fermentation of coffee fruits play an essential role in the metabolites formation that influence the nutritional and sensory quality of the beverage. Thus, the objective of this study was to analyze the effect of coffee cherries processing and fermentation conditions on the microbial communities and the chemical and sensory quality of the beverage. Induced fermentation changed in the bacteria and fungi communities (Treatments: T1, T3, and T7). Klebsiella sp. inoculation (T1) allowed an increase in richness of bacteria and 81 points in the sensory score over the fermentation time. However, there was a reduction in richness of microbial community in treatments with Saccharomyces cerevisiae (T3 and T7). An increase in the indexes of microbial diversity was observed in 72 h in treatment with coffee pulp (T2). In treatment with coffee cherries and spontaneous fermentation (T4) had a higher sensory score than other treatments, indicating a sensory gain from 36 to 72 h. Coffee cherries with superficial disinfection (T5) had a reduction in microbial profile, but did not change the final score of the beverage over the 72 h. In T6 (floaters fruits) was observed an alteration in the fungal community (36–72 h) and the lowest sensory score. The impact of adding water on coffee fermentation was dependent on time (T3 and T7). Furthermore, 5-hydroxymethylfurfural has a positive correlation with the final score of the beverage. Thus, microbial profile and sensory score of beverages are dependent of conditions of processing of coffee fruits and fermentation.

Gastrointestinal microbiota and immune cells interact closely and display regional specificity; however, little is known about how these communities differ with location. Here, we simultaneously assess microbiota and single immune cells across the healthy, adult human colon, with paired characterization of immune cells in the mesenteric lymph nodes, to delineate colonic immune niches at steady state. We describe distinct helper T cell activation and migration profiles along the colon and characterize the transcriptional adaptation trajectory of regulatory T cells between lymphoid tissue and colon. Finally, we show increasing B cell accumulation, clonal expansion and mutational frequency from the cecum to the sigmoid colon and link this to the increasing number of reactive bacterial species. The gut microbiota and their proximate immune cells engage in a dialog of reciprocal regulation. James and colleagues describe how immune cell and microbiotal populations vary along the length of the human colon.

Brazilian shellmounds are archaeological sites with a high concentration of marine faunal remains. There are more than 2000 sites along the coast of Brazil that range in age from 8,720 to 985 cal BP. Here, we studied the ichthyoarchaeological remains (i.e., cranial/postcranial bones, otoliths, and teeth, among others) at 13 shellmounds on the southern coast of the state of Rio de Janeiro, which are located in coastal landscapes, including a sandy plain with coastal lagoons, rocky islands, islets and rocky bays. We identified patterns of similarity between shellmounds based on fish diversity, the ages of the assemblages, littoral geomorphology and prehistoric fisheries. Our new radiocarbon dating, based on otolith samples, was used for fishery characterization over time. A taxonomical study of the ichthyoarchaeological remains includes a diversity of 97 marine species, representing 37% of all modern species (i.e., 265 spp.) that have been documented along the coast of Rio de Janeiro state. This high fish diversity recovered from the shellmounds is clear evidence of well-developed prehistoric fishery activity that targeted sharks, rays and finfishes in a productive area influenced by coastal marine upwelling. The presence of adult and neonate shark, especially oceanic species, is here interpreted as evidence of prehistoric fisheries capacity for exploitation and possibly overexploitation in nursery areas. Various tools and strategies were used to capture finfish in seasonal fisheries, over rocky reef bottoms and in sandy littoral environments. Massive catches of whitemouth croaker, main target dermersal species of South Atlantic coast, show evidence of a reduction in body size of approximately 28% compared with modern fisheries. Fishery activity involving vulnerable species, especially in nursery areas, could mark the beginning of fish depletion along the southeastern Brazilian coast and the collapse of natural fish populations.

The physical or morphological integrity of the coffee bean during post-harvest processing directly influences the economic value and sensory quality of the coffee beverage. Breakdowns in the outer layers of the beans are characteristics observed for the morphological and economic classification of coffee beans during the commercialization of this product. However, physical changes in the inner layers of the beans that are not seen with the naked eye can also influence the sensory quality of the coffee. Therefore, the objective of this study was to relate changes in the physical structure of coffee beans roasted by four different processes (light, medium, dark, and baked) with the sensory attributes of the beverage. The analyses of the physical characteristics of the coffee beans were carried out by X-ray microtomography and the sensory profile was determined using the Specialty Coffee Association of America protocol. The roasting profile with the highest sensory scores showed higher values for total pore space volume and a negative Euler number. However, the roasting profiles that fluctuated between the highest and lowest of scores of the sensory attributes did not present standardized behavior for the connectivity, Euler number, and total pore space volume. Hence, morphological or physical changes in the coffee beans caused by the different types of roasting correlate with changes in the sensorial profile. Furthermore, the sensory discrimination of these coffee beans among the different roast profiles may be observed by the joint analysis of the flavor and fragrance scores.

mNET-seq generates genome-wide, single-nucleotide–resolution data on Pol II occupancy and co-transcriptional RNA processing, with the unique ability to link these processes to Pol II C-terminal domain phosphorylation states. The transcription cycle of RNA polymerase II (Pol II) correlates with changes to the phosphorylation state of its large subunit C-terminal domain (CTD). We recently developed Native Elongation Transcript sequencing using mammalian cells (mNET-seq), which generates single-nucleotide–resolution genome-wide profiles of nascent RNA and co-transcriptional RNA processing that are associated with different CTD phosphorylation states. Here we provide a detailed protocol for mNET-seq. First, Pol II elongation complexes are isolated with specific phospho-CTD antibodies from chromatin solubilized by micrococcal nuclease digestion. Next, RNA derived from within the Pol II complex is size fractionated and Illumina sequenced. Using mNET-seq, we have previously shown that Pol II pauses at both ends of protein-coding genes but with different CTD phosphorylation patterns, and we have also detected phosphorylation at serine 5 (Ser5-P) CTD-specific splicing intermediates and Pol II accumulation over co-transcriptionally spliced exons. With moderate biochemical and bioinformatic skills, mNET-seq can be completed in ∼6 d, not including sequencing and data analysis.

As the first line of defence against pathogens, cells mount an innate immune response, which varies widely from cell to cell. The response must be potent but carefully controlled to avoid self-damage. How these constraints have shaped the evolution of innate immunity remains poorly understood. Here we characterize the innate immune response’s transcriptional divergence between species and variability in expression among cells. Using bulk and single-cell transcriptomics in fibroblasts and mononuclear phagocytes from different species, challenged with immune stimuli, we map the architecture of the innate immune response. Transcriptionally diverging genes, including those that encode cytokines and chemokines, vary across cells and have distinct promoter structures. Conversely, genes that are involved in the regulation of this response, such as those that encode transcription factors and kinases, are conserved between species and display low cell-to-cell variability in expression. We suggest that this expression pattern, which is observed across species and conditions, has evolved as a mechanism for fine-tuned regulation to achieve an effective but balanced response. Comparison of transcriptomic data from immune-stimulated cells across different species sheds light on the architecture of the innate immune response.