Microbial communities such as those residing in the human gut are highly diverse and complex, and many with important implications for health and diseases. The effects and functions of these microbial communities are determined not only by their species compositions and diversities but also by the dynamic intra- and inter-cellular states at the transcriptional level. Powerful and scalable technologies capable of acquiring single-microbe-resolution RNA sequencing information in order to achieve a comprehensive understanding of complex microbial communities together with their hosts are therefore utterly needed. Here we report the development and utilization of a droplet-based smRNA-seq (single-microbe RNA sequencing) method capable of identifying large species varieties in human samples, which we name smRandom-seq2. Together with a triple-module computational pipeline designed for the bacteria and bacteriophage sequencing data by smRandom-seq2 in four human gut samples, we established a single-cell level bacterial transcriptional landscape of human gut microbiome, which included 29,742 single microbes and 329 unique species. Distinct adaptive response states among species in Prevotella and Roseburia genera and intrinsic adaptive strategy heterogeneity in Phascolarctobacterium succinatutens were uncovered. Additionally, we identified hundreds of novel host-phage transcriptional activity associations in the human gut microbiome. Our results indicated that smRandom-seq2 is a high-throughput and high-resolution smRNA-seq technique that is highly adaptable to complex microbial communities in real-world situations and promises new perspectives in the understanding of human microbiomes.
Humans ; Gastrointestinal Microbiome/genetics* ; Bacteriophages/physiology* ; High-Throughput Nucleotide Sequencing ; Sequence Analysis, RNA/methods* ; Bacteria/virology*

ObjectiveTo study the accumulation and absorption of dry matter and nutrients in Curcuma phaeocaulis from Sichuan， the origin of Curcumae Radix （tuber） and Curcumae Rhizoma （rhizome）， to explore the growth and development laws and nutrient demand characteristics of the medicinal species， and thus to lay a theoretical basis for rational fertilization. MethodThe plant growth indexes， dry matter accumulation， and nutrient content of C. phaeocaulis at different growth stages in Sichuan were measured and analyzed. ResultThis medicinal species featured the dominant growth of aboveground leaves and stems before October and growth of underground part （particularly the rhizomes and tubers） from October. During the whole growth period， the accumulation of nitrogen， phosphorus， potassium， calcium， magnesium， iron， manganese， zinc， and copper per plant was 2 450.31， 907.09， 3 171.18， 625.94， 493.38， 14.53， 2.24 ， 2.93， 0.46 mg， respectively， with the order of potassium > nitrogen > phosphorus > calcium > magnesium > iron > zinc > manganese > copper. ConclusionThe species needs sufficient potassium and nitrogen， appropriate amount of phosphorus， calcium， and magnesium， a small amount of iron， and very little zinc， manganese， and copper for growth， and potassium is particularly important. Nitrogen， phosphorus， and potassium fertilizers supply macroelements and nitrogen fertilizer should be supplemented at seedling stage， leafy stage， early rhizome expansion stage， tuber expansion stage， and the second expansion stage of rhizome. Phosphorus and potassium fertilizers should be applied at seedling stage， tuber expansion stage， secondary expansion stage of rhizome， and dry matter accumulation stage of tuber and rhizome. At seedling stage， leafy stage， early rhizome expansion stage， tuber expansion stage， and the second expansion stage of rhizome， a variety of medium and trace elements-containing foliar fertilizers should be used.

The importance of structural variants(SVs)for human phenotypes and diseases is now recognized.Although a variety of SV detection platforms and strategies that vary in sensitivity and specificity have been developed,few benchmarking procedures are available to confidently assess their performances in biological and clinical research.To facilitate the validation and application of these SV detection approaches,we established an Asian reference material by characterizing the genome of an Epstein-Barr virus(EBV)-immortalized B lymphocyte line along with identified benchmark regions and high-confidence SV calls.We established a high-confidence SV callset with 8938 SVs by integrating four alignment-based SV callers,including 109x Pacific Biosciences(PacBio)continuous long reads(CLRs),22 x PacBio circular consensus sequencing(CCS)reads,104x Oxford Nanopore Technologies(ONT)long reads,and 114×Bionano optical mapping plat-form,and one de novo assembly-based SV caller using CCS reads.A total of 544 randomly selected SVs were validated by PCR amplification and Sanger sequencing,demonstrating the robustness of our SV calls.Combining trio-binning-based haplotype assemblies,we established an SV benchmark for identifying false negatives and false positives by constructing the continuous high-confidence regions(CHCRs),which covered 1.46 gigabase pairs(Gb)and 6882 SVs supported by at least one diploid haplotype assembly.Establishing high-confidence SV calls for a benchmark sample that has been characterized by multiple technologies provides a valuable resource for investigating SVs in human biology,disease,and clinical research.

Objective To develop an ultrasmall superparamagnetic iron oxide(USPIO)based MR probe targeting CD40 mutant and investigate its biological and chemical properties and its targeting effect on ovarian cancer cells in vitro.Methods To prepare immunologically competent probe,the monoclonal antibody was conjugated with USPIO particles modified by DMSA based on chemical crosslinking method.The USPIO labeled anti-human CD40 mutant monoclonal antibody 5H6(5H6-USPIO)was the experimental probe,and the USPIO labeled anti-human CD40 monoclonal antibody 5C11(5C11-USPIO)and USPIO served as control agents.The flow cytometry,confocal microscopy and Prussian blue staining were employed to assess the magnetic performance and analyze its bioactivity of the probe.The probe's cell MR imaging in vitro was carried out using ovarian caner cells(HO8910)with high CD40 mutant expression.The analysis of signal data of different groups was conducted by using one-way ANOVA andLSD test.The probe's effect on ovarian caner cells' growth was measured by CCK-8 kit.Results The stable molecular probe carrying nanoparticles and CD40 mutant antibody was built and purified successfully.The probe had similar magnetic property compared with original USPIO.Immunofluorescence and Prussian blue staining confirmed that the molecular probe could recognize CD40 mutant on ovarian cancer cells(HO8910)with high specificity.The probe had no effect on the growth of HO8910 cells.MR cell imaging in vitro showed that the value of T2 and T2 * decreased significantly after the probe binding with HO8910 cells and T2WI became darker than control groups.The T2 and T2* relaxation time of 5H6-USPIO group was(40.05 ± 1.62)ms and(3.08 ± 0.11)ms,respectively.The T2 and T2 * relaxation time of 5H6-USPIO group was shorter than 5C11-USPIO [(85.38 ± 4.74)and(11.82 ± 1.00)ms,respectively]and USPIO[(91.62 ± 3.35)and(13.60 ± 1.92)ms,respectively]groups with statistical significance(F =196.29,60.73,P ＜0.01).The difference of T2 and T2* relaxation time between 5C11-USPIO and USPIO groups showed no statistical significance (P ＞0.05).Conclusions CD40 mutant antibody can conjugate with ultrasmall superparamagnetic iron oxide particles to form MR molecular probe using chemical crosslinking method.The probe has good magnetic characters and high bioactivity.The probe can specifically bind with HO8910 cells.